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Sample records for stably transfected bioluminescent

  1. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  2. Stably luminescent Staphylococcus aureus clinical strains for use in bioluminescent imaging.

    PubMed

    Plaut, Roger D; Mocca, Christopher P; Prabhakara, Ranjani; Merkel, Tod J; Stibitz, Scott

    2013-01-01

    In vivo bioluminescent imaging permits the visualization of bacteria in live animals, allowing researchers to monitor, both temporally and spatially, the progression of infection in each animal. We sought to engineer stably luminescent clinical strains of Staphylococcus aureus, with the goal of using such strains in mouse models. The gram-positive shuttle vector pMAD was used as the backbone for an integration plasmid. A chloramphenicol resistance gene, a modified lux operon from Photorhabdus luminescens, and approximately 650 bp of homology to the chromosome of the USA300 S. aureus strain NRS384 were added, generating plasmid pRP1195. Electroporation into strain RN4220 followed by temperature shift led to integration of pRP1195 into the chromosome. The integrated plasmid was transferred to clinical strains by phage transduction. Luminescent strains displayed no in vitro growth defects. Moreover, luminescence was stable in vitro after three rounds of subculture over 48 hours of growth in the absence of antibiotics. Mice were infected with a luminescent strain of NRS384 in skin and intravenous models. In a mouse skin model, luminescent bacteria were present in lesions that formed and cleared over the course of several days, and in an intravenous model, bacteria inoculated in the mouse tail vein were observed spreading to multiple tissues. No statistically significant difference in virulence was observed between NRS384 and the luminescent strain in either infection model. These preliminary data suggest that this luminescent USA300 strain is suitable for use in mouse models. Similar strains were engineered using other sequenced clinical strains. Because these strains are stably luminescent, they should prove useful in animal models of infection.

  3. Characterization of the human CUTA isoform2 present in the stably transfected HeLa cells.

    PubMed

    Yang, Jingchun; Yang, Huirong; Yan, Lichong; Yang, Liu; Yu, Long

    2009-01-01

    CUTA, Homo sapiens divalent cation tolerance homolog, has been implicated in anchoring of acetylcholinesterase in neuronal cell membranes. However, a protein highly homologous to CUTA in Rattus norvegicus is structurally similar to the signal transduction protein PII, and this similarity suggests an intriguing role of CUTA in signal transduction. Recent researches indicated that CUTA was one of the 35 key genes responsible for lactation in mammary gland development. However, the physiological role of CUTA is still unclear, so more information of this gene is needed. In this study, the expression profile of CUTA gene in human tissues was examined, and our research revealed that CUTA gene was constitutively expressed in all of the 18 tissues tested. As reported, CUTA gene has five variant transcripts encoding three isoforms with different N terminals. CUTA isoform2 is encoded by three of the five variant transcripts as the common part of the three isoforms. So CUTA isoform2 was chose as representative to characterize the CUTA protein. We constructed a HeLa cell line stably transfected with the encoding sequence of CUTA isoform2 for further study. The subcellular location and oligomeric structure of the CUTA isoform2 was analyzed in the stable cell lines. It was found that the CUTA isoform2 was mainly located in mitochondria as a new potential mitochondrial protein. Furthermore, CUTA isoform2 formed trimers in cell lysate with the possible occurrence of heteropolymers. These findings would be helpful to the further study on the specific function of CUTA protein.

  4. Synthesis of recombinant human procollagen II in a stably transfected tumour cell line (HT1080).

    PubMed Central

    Fertala, A; Sieron, A L; Ganguly, A; Li, S W; Ala-Kokko, L; Anumula, K R; Prockop, D J

    1994-01-01

    -number-dependent expression of an exogenous collagen gene in stably transfected cells. Images Figure 2 Figure 4 PMID:8129728

  5. Stably transfected human cell lines as fluorescent screening assay for nuclear factor KB activation dependent gene expression

    NASA Astrophysics Data System (ADS)

    Hellweg, Christine E.; Baumstark-Khan, Christa; Horneck, Gerda

    2004-06-01

    Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. For identifying conditions which are capable to modify this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter proteins Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) has been developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing four copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP / d2EGFP expression in up to 90 % of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB dependent gene expression. The time course of d2EGFP expression after treatment with TNF-alpha or phorbol ester was measured using flow cytometry. Cellular response to TNF-alpha was faster than to phorbol ester. Treatment of cells with TNF-alpha and DMSO revealed antagonistic interactions of these substances in the activation NF-kappaB dependent gene expression. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.

  6. THE EVALUATION OF PEPTIDE/HISTIDINE TRANSPORTER 1 (PHT1) FUNCTION: UPTAKE KINETICS UTILIZING A COS-7 STABLY TRANSFECTED CELL LINE.

    PubMed

    Lindley, David J; Carl, Stephen M; Mowery, Stephanie A; Knipp, Gregory T

    2011-10-01

    There have been relatively few studies focused on the proton-dependent oligopeptide transporter (POT) superfamily member, Peptide/Histidine Transporter 1 (PHT1), with respect to its contribution to the ADME of peptides and peptide-based drugs. These studies were conducted to determine hPHT1-mediated, H(+)-dependent uptake kinetics of histidine, carnosine, Gly-Sar and valacyclovir in stably transfected hPHT1-COS-7 cells comparative to kinetics determined in an empty vector (Mock) stably transfected cell line. The results suggest that Gly-Sar appears to be a substrate for PHT1 based on efflux from the stably transfected hPHT1 COS-7 cells. Histidine and Gly-Sar concentration- and time-dependent studies suggest mixed-uptake kinetics. These studies suggest that stably transfected hPHT1-COS-7 cells exhibit different uptake kinetics than those observed in our previous studies and illustrate the requirement for experiments to delineate the physiological role of hPHT1.

  7. Stably transfected human cells overexpressing rat brain endopeptidase 3.4.24.16: biochemical characterization of the activity and expression of soluble and membrane-associated counterparts.

    PubMed

    Vincent, B; Dauch, P; Vincent, J P; Checler, F

    1997-02-01

    We recently cloned endopeptidase-24.16 (neurolysin; EC 3.4.24.16), a neurotensin-degrading peptidase likely involved in the physiological termination of the neurotensinergic signal in the central nervous system and in the gastrointestinal tract. We stably transfected human kidney cells with the pcDNA3-lambda 7aB1 construction bearing the whole open reading frame encoding the rat brain peptidase. Transfectants displayed endopeptidase-24.16 immunoreactivity and exhibited QFS- and neurotensin-hydrolyzing activities, the biochemical and specificity properties of which fully matched those observed with the purified murine enzyme. Cryoprotection experiments and substrate degradation by intact plated cells indicated that transfectants exhibited a membrane-associated form of endopeptidase-24.16, the catalytic site of which clearly faced the extracellular domain. Transfected cells were unable to secrete the enzyme. Overall, our experiments indicate that we have obtained stably transfectant cells that overexpress an enzymatic activity displaying biochemical properties identical to those of purified endopeptidase-24.16. The membrane-associated counterpart and lack of secretion of the enzyme were clearly reminiscent of what was observed with pure cultured neurons, but not with astrocytes. Therefore, the transfected cell model described here could prove useful for establishing, by a mutagenesis approach, the structural elements responsible for the "neuronal" phenotype exhibited by the enzyme in transfected cells.

  8. The influence of hypoxia on bioluminescence in luciferase-transfected gliosarcoma tumor cells in vitro.

    PubMed

    Moriyama, Eduardo H; Niedre, Mark J; Jarvi, Mark T; Mocanu, Joseph D; Moriyama, Yumi; Subarsky, Patrick; Li, Buhong; Lilge, Lothar D; Wilson, Brian C

    2008-06-01

    Firefly luciferase catalyzes the emission of light from luciferin in the presence of oxygen and adenosine triphosphate. This bioluminescence is commonly employed in imaging mode to monitor tumor growth and treatment responses in vivo. A potential concern is that, since solid tumors are often hypoxic, either constitutively and/or as a result of treatment, the oxygen available for the bioluminescence reaction could be reduced to limiting levels, leading to underestimation of the actual number of luciferase-labeled cells during in vivo experiments. We present studies of the oxygen dependence of bioluminescence in vitro in rat 9 L gliosarcoma cells tagged with the firefly luciferase gene (9L(luc)). We demonstrate that the bioluminescence signal decreases at pO(2) bioluminescence reaction and so is responsible for the reduction of bioluminescence signal in 9L(luc) cells in acute hypoxia, rather than luciferase expression or oxygen itself.

  9. Bioluminescence.

    ERIC Educational Resources Information Center

    Jones, M. Gail

    1993-01-01

    Describes bioluminescence and the chemistry of how it occurs. Presents information for conducting the following classroom activities: (1) firefly mimic; (2) modeling deep-sea fish; (3) sea fireflies; and (4) the chemistry of light. (PR)

  10. [Construction of mouse VCAM-1 expression vector and establishment of stably transfected MSC line C3H10T1/2].

    PubMed

    Chen, Hui; Zhu, Heng; Chu, Ya-Nan; Xu, Fen-Fen; Liu, Yuan-Lin; Tang, Bo; Li, Xi-Mei; Hu, Liang-Ding; Zhang, Yi

    2014-10-01

    This study was aimed to construct the mouse VCAM-1 expression vector, to establish the stably transfected MSC line and to investigate the effect of VCAM-1-modified mesenchymal stem cells (MSC) on the immunological characteristics of MSC. The cDNA of murine VCAM-1 gene was amplified by RT-PCR from the total RNA isolated from the mouse spleen; then the cDNA was inserted into the retrovirus vector PMSCVmigr-1; the recombinant plasmid was confirmed by restriction endonuclease experiments and sequencing, then designated as PMSCVmigr-1-mVCAM-1; the recombinant plasmid PMSCVmigr-1-mVCAM-1 was transfected into 293 cells by lipofecamin and the supernatant was collected to transfect MSC cell line (C3H10T1/2). Moreover, VCAM-1 expression on MSC was evaluated by FACS. Furthermore, the inhibitory effect of VCAM-1-MSC on lymphocytic transformation was tested by (3)H-TdR incorporation assay. The results indicated that the successful construction of recombinant retroviral expression plasmid of mouse VCAM-1 was confirmed by digesting and sequancing. After transfection of MSC with retroviral supernaptant, the high expression of VCAM-1 on MSC could be detected by flow cytometry. The MSC high expressing VCAM-1 could significantly inhibit the proliferation of Con A-inducing lymphocytes in dose-depentent marrer. It is concluded that recombinant retroviral encoding VCAM-1 (PMSCVmigr-1-mVCAM-1) has been successfully constructed and mouse VCAM-1 has been stably expressed in C3H10T1/2. MSC over-expressing VCAM-1 show more potent immunosuppressive effect on cellular immune reaction in vitro. Our data laid a foundation for the subsequent studying the effect of VCAM-1 transfecting into MSC on immune related disease study.

  11. Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line.

    PubMed

    Legler, J; van den Brink, C E; Brouwer, A; Murk, A J; van der Saag, P T; Vethaak, A D; van der Burg, B

    1999-03-01

    Development of an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression (ER-CALUX) assay was attempted by stable transfection of luciferase reporter genes in a number of cell lines. Stable transfection of the chimeric Gal4 estrogen receptor and luciferase gene constructs in MCF-7 breast cancer and Hepa.1c1c7 mouse hepatoma cell lines, as well as transfection of a newly constructed luciferase reporter gene pEREtata-Luc in the ECC-1 human endometrial cell line, resulted in constitutive, non-estradiol-inducible clones. Stable transfection of pEREtata-Luc in the T47D breast cancer cell line, however, resulted in an extremely sensitive, highly responsive cell line. Following a 24-h exposure to estradiol (E2), stably transfected T47D.Luc cells demonstrated a detection limit of 0.5 pM, an EC50 of 6 pM, and a maximum induction of 100-fold relative to solvent controls. No clear reduction in responsiveness has been found over extended culture periods (50 passages). Anti-estrogens ICI 182,780, TCDD, and tamoxifen inhibited the estradiol-mediated luciferase induction. Genistein, nonylphenol, and o,p'DDT were the most potent (pseudo-)estrogens tested in this system (EC50 100, 260, and 660 nM, respectively). Determination of interactive effects of the (pseudo-)estrogens nonylphenol, o,p'DDT, chlordane, endosulfan, dieldrin, and methoxychlor revealed that, in combination with 3 pM E2, (pseudo-)estrogens were additive. Slightly more than additive effects (less than 2-fold) were found for combinations of dieldrin and endosulfan tested in the range of 3 to 6 microM. At these concentrations, the combination of endosulfan and chlordane demonstrated additive interaction. The ER-CALUX assay with T47D cells can provide a sensitive, responsive, and rapid in vitro system to detect and measure substances with potential (anti-)estrogenic activity.

  12. Generation and preclinical immunogenicity study of dengue type 2 virus-like particles derived from stably transfected mosquito cells.

    PubMed

    Suphatrakul, Amporn; Yasanga, Thippawan; Keelapang, Poonsook; Sriburi, Rungtawan; Roytrakul, Thaneeya; Pulmanausahakul, Rojjanaporn; Utaipat, Utaiwan; Kawilapan, Yanee; Puttikhunt, Chunya; Kasinrerk, Watchara; Yoksan, Sutee; Auewarakul, Prasert; Malasit, Prida; Charoensri, Nicha; Sittisombut, Nopporn

    2015-10-13

    Recent phase IIb/III trials of a tetravalent live attenuated vaccine candidate revealed a need for improvement in the stimulation of protective immunity against diseases caused by dengue type 2 virus (DENV-2). Our attempts to develop particulate antigens for possibly supplementing live attenuated virus preparation involve generation and purification of recombinant DENV-2 virus-like particles (VLPs) derived from stably (prM+E)-expressing mosquito cells. Two VLP preparations generated with either negligible or enhanced prM cleavage exhibited different proportions of spherical particles and tubular particles of variable lengths. In BALB/c mice, VLPs were moderately immunogenic, requiring adjuvants for the induction of strong virus neutralizing antibody responses. VLPs with enhanced prM cleavage induced higher levels of neutralizing antibody than those without, but the stimulatory activity of both VLPs was similar in the presence of adjuvants. Comparison of EDIII-binding antibodies in mice following two adjuvanted doses of these VLPs revealed subtle differences in the stimulation of anti-EDIII binding antibodies. In cynomolgus macaques, VLPs with enhanced prM cleavage augmented strongly neutralizing antibody and EDIII-binding antibody responses in live attenuated virus-primed recipients, suggesting that these DENV-2 VLPs may be useful as the boosting antigen in prime-boost immunization. As the levels of neutralizing antibody induced in macaques with the prime-boost immunization were comparable to those infected with wild type virus, this virus-prime VLP-boost regimen may provide an immunization platform in which a need for robust neutralizing antibody response in the protection against DENV-2-associated illnesses could be tested.

  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. The Induction of Serine/Threonine Protein Phosphorylations by a PDGFR/TrkA Chimera in Stably Transfected PC12 Cells*

    PubMed Central

    Biarc, Jordane; Chalkley, Robert J.; Burlingame, A. L.; Bradshaw, Ralph A.

    2012-01-01

    Stably transfected PC12 cells expressing a chimeric receptor composed of the extracellular domain of the platelet-derived growth factor receptor BB and the transmembrane and intracellular domains of TrkA, the nerve growth factor receptor, were stimulated for 20 min with platelet-derived growth factor and the resulting phosphoproteome was determined from affinity purified tryptic peptides identified by tandem MS (MS/MS) analyses. The changes in the levels of individual phosphorylation sites in stimulated cells versus control were ascertained by the stable isotope labeling of amino acids in cell culture technique. A total of 2035 peptides (806 proteins) were indentified and quantified in both data sets. Of these, 424 phosphopeptides on 259 proteins were found to be up-regulated and 392 sites on 206 proteins were down-regulated (1.8-fold or more). Protein kinases and phosphatases, as well as sites in many proteins involved in G-protein signaling, were prominently represented in the up-regulated group and more than half of the kinase up-regulated phosphosites could be clustered into three sequence motifs; a similar distribution was also found for the down-regulated sites. A comparison of the up-regulated motif profile observed to that calculated from a previous study of the EGFR-induced phosphoproteome in human HeLa cells at the same time point showed a considerable amount of similarity, supporting the view that RTK signal transduction pathways and downstream modifications are likely to be extensively overlapping. PMID:22027198

  15. Development of stably transfected human and rat hepatoma cell lines for the species-specific assessment of xenobiotic response enhancer module (XREM)-dependent induction of drug metabolism.

    PubMed

    Fery, Yvonne; Mueller, Stefan O; Schrenk, Dieter

    2010-11-09

    Based on our current knowledge, PXR holds a key position in the induction of a selective battery of enzymes and transporters of drug metabolism. In order to prevent serious adverse drug effects or unpredicted drug-drug interactions (DDI), it is compulsory to investigate the possible inducing potency of drugs under development. Furthermore, analysis of the inducing potency of environmental pollutants and new or manufactured chemicals is part of toxicological risk assessment. In non-transfected human HepG2 and rat H4IIE hepatoma cells, we examined the characteristics of expression of 45 genes involved in drug metabolism. A few gene products such as CYP2B6 or CYP3A4 mRNA were prominent in HepG2 cells while their major rat counterparts were, e.g., CYP2B3 or CYP3A1/3A3. Furthermore, a number of xenobiotic receptors including PXR were expressed in both cell lines. A number of genes were regulated in a cell type and species-specific manner after incubation with the prototypical PXR agonists rifampicin or dexamethasone, respectively. Then, we established cell-based reporter gene assays for screening for PXR-dependent induction of drug metabolism. HepG2 and H4IIE cells were stably transfected with a reporter gene containing PXR responsive elements (XREMs) which mediate the induction of PXR target genes such as CYP3A enzymes. With both stable cell lines the CYP inducers clotrimazole, dexamethasone, omeprazole, phenobarbital, rifampicin, as well as the drug candidate EMD 392949 and the brominated flame retardants hexabromocylododecane (HBCD) and a pentabromodiphenyl ether (pentaBDE) mixture were screened. In the human HepG2-XREM3 and rat H4IIE-XREM3 cells, clotrimazole and HBCD were found as common activators of the human and rat PXR whereas pentaBDE was more effective with the human cell system. Omeprazole and phenobarbital did not induce the rat PXR-dependent reporter gene expression in H4IIE-XREM3 cells, while a moderate increase was found in HepG2-XREM3 cells. EMD 392949

  16. Intracellular localization of the PDE4A cAMP-specific phosphodiesterase splice variant RD1 (RNPDE4A1A) in stably transfected human thyroid carcinoma FTC cell lines.

    PubMed Central

    Pooley, L; Shakur, Y; Rena, G; Houslay, M D

    1997-01-01

    Cells of two human follicular thyroid carcinoma cell lines (FTC133, FTC236) were stably transfected with a cDNA encoding the PDE4A cAMP-specific phosphodiesterase (PDE) splice variant RD1 (RNPDE4A1A) so as to generate the cloned cell lines, FTC133A and FTC236A. This allowed the expression of a novel rolipram-inhibited cAMP-specific PDE activity in these cells. Unlike the parent cell lines in which Ca2+/calmodulin caused a profound activation (approx. 3-4-fold) of homogenate PDE activity, no such stimulation was evident in the RD1-expressing cell lines, indicating loss of PDE1 activity. Reverse transcriptase-PCR analysis indicated that this was due to the down-regulation of the PDE1C isoform. The novel PDE4 activity in transfected cells was located exclusively in the membrane fraction, as was immunoreactive RD1. Low concentrations of the detergent Triton X-100, but not high NaCl concentrations, allowed RD1 to be solubilized. Laser scanning confocal immunofluorescence analyses identified RD1 immunoreactivity in a discrete perinuclear region of these RD1-expressing transfected cell lines. A similar pattern of labelling was observed using the antiserum Tex1, which specifically identified the Golgi apparatus. Treatment of FTC133A cells with the Golgi-perturbing agents monensin and brefeldin A led to a similar redistribution of immunoreactive species detected using both the Tex1 and anti-RD1 antisera. It is suggested that the PDE4A splice variant RD1 contains a membrane-association signal which allows the targeted expression of RD1 within the Golgi complex of these human follicular thyroid carcinoma cell lines. PMID:9003417

  17. Cytotoxicity of 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT) and analogues in wild type and CYP3A4 stably transfected HepG2 cells.

    PubMed

    Frederick, Douglas M; Jacinto, Erina Y; Patel, Niti N; Rushmore, Thomas H; Tchao, Ruy; Harvison, Peter J

    2011-12-01

    The thiazolidinedione (TZD) ring is a constituent of the glitazones that are used to treat type II diabetes. Liver injury has been reported following chronic glitazone use; however, they do not produce hepatic damage in common laboratory animal species. In contrast, 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT) causes hepatotoxicity in rats. DCPT toxicity is dependent upon the presence of an intact TZD ring and cytochrome P450 (CYP)-mediated biotransformation. To further investigate TZD ring-induced toxicity, DCPT and several structural analogues or potential metabolites were tested in vitro using wild type human hepatoma HepG2 and HepG2 cells stably transfected with the CYP3A4 isozyme. CYP3A4 activity was confirmed by measuring testosterone 6β-hydroxylation. Both cell lines were treated with 0-250 μM of the compounds in Hanks' balanced salt solution. Cell viability was measured after 24 h. DCPT and S-(3,5-dichlorophenyl)aminocarbonyl thioglycolic acid (DCTA) were the most toxic compounds of the series. Furthermore, DCPT was significantly more toxic in transfected cells (LC50=160.2±5.9 μM) than in wild type cells (LC50=233.0±19.7 μM). Treatment with a CYP3A4 inhibitor or inducer attenuated or potentiated DCPT cytotoxicity, respectively. These results suggest that DCPT-induced cytotoxicity in the transfected HepG2 cells is partially dependent on CYP3A4.

  18. Cytotoxicity of 3-(3,5-Dichlorophenyl)-2,4-thiazolidinedione (DCPT) and Analogues in Wild Type and CYP3A4 Stably Transfected HepG2 Cells

    PubMed Central

    Frederick, Douglas M.; Jacinto, Erina Y.; Patel, Niti N.; Rushmore, Thomas H.; Tchao, Ruy; Harvison, Peter J.

    2011-01-01

    The thiazolidinedione (TZD) ring is a constituent of the glitazones that are used to treat type II diabetes. Liver injury has been reported following chronic glitazone use; however, they do not produce hepatic damage in common laboratory animal species. In contrast, 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT) causes hepatotoxicity in rats. DCPT toxicity is dependent upon the presence of an intact TZD ring and cytochrome P450 (CYP)-mediated biotransformation. To further investigate TZD ring-induced toxicity, DCPT and several structural analogues or potential metabolites were tested in vitro using wild type human hepatoma HepG2 and HepG2 cells stably transfected with the CYP3A4 isozyme. CYP3A4 activity was confirmed by measuring testosterone 6β-hydroxylation. Both cell lines were treated with 0-250 μM of the compounds in Hanks' balanced salt solution. Cell viability was measured after 24 hrs. DCPT and S-(3,5-dichlorophenyl)aminocarbonyl thioglycolic acid (DCTA) were the most toxic compounds of the series. Furthermore, DCPT was significantly more toxic in transfected cells (LC50 = 160.2 ± 5.9 μM) than in wild type cells (LC50 = 233.0 ± 19.7 μM). Treatment with a CYP3A4 inhibitor or inducer attenuated or potentiated DCPT cytotoxicity, respectively. These results suggest that DCPT-induced cytotoxicity in the transfected HepG2 cells is partially dependent on CYP3A4. PMID:21964476

  19. Evaluation of in vitro screening system for estrogenicity: comparison of stably transfected human estrogen receptor-α transcriptional activation (OECD TG455) assay and estrogen receptor (ER) binding assay.

    PubMed

    Lee, Hae Kyung; Kim, Tae Sung; Kim, Chang Yeong; Kang, Il Hyun; Kim, Mi Gyeong; Jung, Ki Kyung; Kim, Hyung Sik; Han, Soon Young; Yoon, Hae Jung; Rhee, Gyu Seek

    2012-01-01

    The estrogenic activity of industrial chemicals, di(2-ethylhexyl) phthalate (DEHP), di(n-butyl) phthalate (DBP), benzylbutyl phthalate (BBP), diethyl phthalate (DEP), tetrabromobisphenol A (TBBPA), bisphenol A (BPA), and nonylphenol (NP), was compared using OECD test guideline 455(TG455), stably transfected transcriptional activation (STTA) and estrogen receptor (ER) binding assays. The estrogenic activity of BBP, BPA and NP were approximately 180,000-fold (PC(50), 4.32 x 10(-6 )M), 5,000-fold (PC(50), 1.26 x 10(-7) M) and 120,000-fold (PC(50), 2.92 x 10(-6 )M) less than 17β-estradiol (PC(50), 2.43 x 10(-11)M), whereas DEHP, DBP and DEP did not show any estrogenicity activity in the STTA assay. Moreover, binding affinities to human ERα of BBP, BPA, and NP were approximately 200,000-fold (IC(50), 4.91 x 10(-4) M), 8000-fold (IC(50), 1.92 x 10(-5) M) and 1400-fold (IC(50), 3.34 x 10(-6) M) less than 17β-estradiol (IC(50), 2.45 x 10(-9) M) in competitive human ERα binding assay. The relative potencies of STTA assay were very similar to ER binding, E-screen, and Yeast screening assays. Therefore, our results suggested that OECD test guideline TG455 may be useful as a screening test for potential endocrine disruptors.

  20. COL1A1 transgene expression in stably transfected osteoblastic cells. Relative contributions of first intron, 3'-flanking sequences, and sequences derived from the body of the human COL1A1 minigene

    NASA Technical Reports Server (NTRS)

    Breault, D. T.; Lichtler, A. C.; Rowe, D. W.

    1997-01-01

    Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3'-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.

  1. Flavoprotein miniSOG Cytotoxisity Can Be Induced By Bioluminescence Resonance Energy Transfer

    PubMed Central

    Shramova, E.I.; Proshkina, G.M.; Chumakov, S.P.; Khodarovich, Yu.M.; Deyev, S.M.

    2016-01-01

    In this study, we investigated the possibility of phototoxic flavoprotein miniSOG (photosensitizer) excitation in cancer cells by bioluminescence occurring when luciferase NanoLuc oxidizes its substrate, furimazine. We have shown that the phototoxic flavoprotein miniSOG expressed in eukaryotic cells in fusion with NanoLuc luciferase is activated in the presence of its substrate, furimazine. Upon such condition, miniSOG possesses photoinduced cytotoxicity and causes a 48% cell death level in a stably transfected cell line. PMID:28050273

  2. Destabilized bioluminescent proteins

    DOEpatents

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  3. Monitoring and quantitative assessment of tumor burden using in vivo bioluminescence imaging

    NASA Astrophysics Data System (ADS)

    Chen, Chia-Chi; Hwang, Jeng-Jong; Ting, Gann; Tseng, Yun-Long; Wang, Shyh-Jen; Whang-Peng, Jaqueline

    2007-02-01

    In vivo bioluminescence imaging (BLI) is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating tumor growth. In this study, the kinetic of tumor growth has been assessed in C26 colon carcinoma bearing BALB/c mouse model. The ability of BLI to noninvasively quantitate the growth of subcutaneous tumors transplanted with C26 cells genetically engineered to stably express firefly luciferase and herpes simplex virus type-1 thymidine kinase (C26/ tk-luc). A good correlation ( R2=0.998) of photon emission to the cell number was found in vitro. Tumor burden and tumor volume were monitored in vivo over time by quantitation of photon emission using Xenogen IVIS 50 and standard external caliper measurement, respectively. At various time intervals, tumor-bearing mice were imaged to determine the correlation of in vivo BLI to tumor volume. However, a correlation of BLI to tumor volume was observed when tumor volume was smaller than 1000 mm 3 ( R2=0.907). γ Scintigraphy combined with [ 131I]FIAU was another imaging modality used for verifying the previous results. In conclusion, this study showed that bioluminescence imaging is a powerful and quantitative tool for the direct assay to monitor tumor growth in vivo. The dual reporter genes transfected tumor-bearing animal model can be applied in the evaluation of the efficacy of new developed anti-cancer drugs.

  4. Expression of a Humanized Viral 2A-Mediated lux Operon Efficiently Generates Autonomous Bioluminescence in Human Cells

    PubMed Central

    Xu, Tingting; Ripp, Steven; Sayler, Gary S.; Close, Dan M.

    2014-01-01

    Background Expression of autonomous bioluminescence from human cells was previously reported to be impossible, suggesting that all bioluminescent-based mammalian reporter systems must therefore require application of a potentially influential chemical substrate. While this was disproven when the bacterial luciferase (lux) cassette was demonstrated to function in a human cell, its expression required multiple genetic constructs, was functional in only a single cell type, and generated a significantly reduced signal compared to substrate-requiring systems. Here we investigate the use of a humanized, viral 2A-linked lux genetic architecture for the efficient introduction of an autobioluminescent phenotype across a variety of human cell lines. Methodology/Principal Findings The lux cassette was codon optimized and assembled into a synthetic human expression operon using viral 2A elements as linker regions. Human kidney, breast cancer, and colorectal cancer cell lines were both transiently and stably transfected with the humanized operon and the resulting autobioluminescent phenotype was evaluated using common imaging instrumentation. Autobioluminescent cells were screened for cytotoxic effects resulting from lux expression and their utility as bioreporters was evaluated through the demonstration of repeated monitoring of single populations over a prolonged period using both a modified E-SCREEN assay for estrogen detection and a classical cytotoxic compound detection assay for the antibiotic Zeocin. Furthermore, the use of self-directed bioluminescent initiation in response to target detection was assessed to determine its amenability towards deployment as fully autonomous sensors. In all cases, bioluminescent measurements were supported with traditional genetic and transcriptomic evaluations. Conclusions/Significance Our results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines

  5. Marine Bioluminescence: Mechanisms and Evaluation

    DTIC Science & Technology

    1999-09-30

    bioluminescent systems Gelatinous zooplankton - Analysis of bioluminescence spectra as a function of depth of occurrence was completed and published (Haddock...attraction of zooplankton predators we have used a new profiling bioluminescence detector system in an examination of the fine scale bioluminescence...bioluminescent systems. J. Mol. Evolution 19, 309-321. PUBLICATIONS Haddock, S.H.D. (1999): Bioluminescent spectra of shallow and deep-sea gelatinous

  6. Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents

    PubMed Central

    Shabani, Mahdi; Hemmati, Sheyda; Hadavi, Reza; Amirghofran, Zahra; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Shokri, Fazel

    2010-01-01

    Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production. PMID:23408356

  7. Laser-induced bioluminescence

    SciTech Connect

    Hickman, G.D.; Lynch, R.V. III

    1981-01-01

    A project has been initiated to determine the feasibility of developing a complete airborne remote sensing system for rapidly mapping high concentration patches of bioluminescent organisms in the world's oceans. Conceptually, this system would be composed of a laser illuminator to induce bioluminescence and a low light level image intensifier for detection of light. Initial laboratory measurements consisted of using a 2-J flash lamp pulsed optical dye laser to excite bioluminescence in the marine dinoflagellate Pyrocustis lunula at ambient temperature using Rhodamine 6G as the lasing dye (585 nm) and a laser pulse width of 1 microsec. After a latency period of 15-20 msec, the bioluminescence maximum occurred in the blue (480 nm is the wavelength maximum for most dinoflagellate bioluminescence) with the peaking occurring approximately 65 msec after the laser pulse. Planned experiments will investigate the effect of different excitation wavelengths and energies at various temperatures and salinities of the cultures.

  8. A multi-phase level set framework for source reconstruction in bioluminescence tomography

    SciTech Connect

    Huang Heyu; Qu Xiaochao; Liang Jimin; He Xiaowei; Chen Xueli; Yang Da'an; Tian Jie

    2010-07-01

    We propose a novel multi-phase level set algorithm for solving the inverse problem of bioluminescence tomography. The distribution of unknown interior source is considered as piecewise constant and represented by using multiple level set functions. The localization of interior bioluminescence source is implemented by tracing the evolution of level set function. An alternate search scheme is incorporated to ensure the global optimal of reconstruction. Both numerical and physical experiments are performed to evaluate the developed level set reconstruction method. Reconstruction results show that the proposed method can stably resolve the interior source of bioluminescence tomography.

  9. Bioluminescence in the Sea

    NASA Astrophysics Data System (ADS)

    Haddock, Steven H. D.; Moline, Mark A.; Case, James F.

    2010-01-01

    Bioluminescence spans all oceanic dimensions and has evolved many times—from bacteria to fish—to powerfully influence behavioral and ecosystem dynamics. New methods and technology have brought great advances in understanding of the molecular basis of bioluminescence, its physiological control, and its significance in marine communities. Novel tools derived from understanding the chemistry of natural light-producing molecules have led to countless valuable applications, culminating recently in a related Nobel Prize. Marine organisms utilize bioluminescence for vital functions ranging from defense to reproduction. To understand these interactions and the distributions of luminous organisms, new instruments and platforms allow observations on individual to oceanographic scales. This review explores recent advances, including the chemical and molecular, phylogenetic and functional, community and oceanographic aspects of bioluminescence.

  10. Impact of Anesthesia Protocols on In Vivo Bioluminescent Bacteria Imaging Results.

    PubMed

    Chuzel, Thomas; Sanchez, Violette; Vandamme, Marc; Martin, Stéphane; Flety, Odile; Pager, Aurélie; Chabanel, Christophe; Magnier, Luc; Foskolos, Marie; Petit, Océane; Rokbi, Bachra; Chereul, Emmanuel

    2015-01-01

    Infectious murine models greatly benefit from optical imaging using bioluminescent bacteria to non-invasively and repeatedly follow in vivo bacterial infection. In this context, one of the most critical parameters is the bioluminescence sensitivity to reliably detect the smallest number of bacteria. Another critical point is the anesthetic approaches that have been demonstrated to impact the bioluminescence flux emission in studies with luciferase-transfected tumor cells. However, this impact has never been assessed on bacteria bioluminescent models. To this end, we investigated the effects of four anesthesia protocols on the bioluminescence flux in a central venous catheter murine model (SKH1-hr(hr) mice) infected by a bioluminescent S. aureus Xen36 strain. Bioluminescence imaging was performed on mice anesthetized by either ketamine/xylazine (with or without oxygen supplementation), or isoflurane carried with air or oxygen. Total flux emission was determined in vivo daily for 3 days and ex vivo at the end of the study together with a CFU counting of the biofilm in the catheter. Bioluminescence flux differences appear between the different anesthetic protocols. Using a ketamine/xylazine anesthesia (with air), bacteria detection was impossible since the bioluminescence signal remains in the background signal. Mice anesthetized with isoflurane and oxygen led to a signal significantly higher to the background all along the kinetics. The use of isoflurane in air presents a bioluminescence signal similar to the use of ketamine/xylazine with oxygen. These data highlight the importance of oxygen to improve bioluminescence flux by bacteria with isoflurane as well as with ketamine/xylazine anesthetics. As a conclusion, we recommend the use of isoflurane anesthetic with oxygen to increase the bioluminescence sensitivity in this kind of study.

  11. Bioluminescent bioreporter integrated circuit

    DOEpatents

    Simpson, Michael L.; Sayler, Gary S.; Paulus, Michael J.

    2000-01-01

    Disclosed are monolithic bioelectronic devices comprising a bioreporter and an OASIC. These bioluminescent bioreporter integrated circuit are useful in detecting substances such as pollutants, explosives, and heavy-metals residing in inhospitable areas such as groundwater, industrial process vessels, and battlefields. Also disclosed are methods and apparatus for environmental pollutant detection, oil exploration, drug discovery, industrial process control, and hazardous chemical monitoring.

  12. A Prototype Bioluminescence Photometer

    DTIC Science & Technology

    1982-02-01

    Seliger et al., 1962) provided maximum mechanical stimulation to dino - flagellates , and incorporated light baffles to permit continuous measurement...was deployed in a shallow coastal area relatively free of any mixing. The principle bioluminescent plankton were the dino - flagellates , as evidenced

  13. Theoretical Study of Dinoflagellate Bioluminescence.

    PubMed

    Wang, Ming-Yu; Liu, Ya-Jun

    2017-03-01

    Dinoflagellates are the most ubiquitous luminescent protists in the marine environment and have drawn much attention for their crucial roles in marine ecosystems. Dinoflagellate bioluminescence has been applied in underwater target detection. The luminescent system of dinoflagellates is a typical luciferin-luciferase one. However, the excited-state oxyluciferin is not the light emitter of dinoflagellate bioluminescence as in most luciferin-luciferase bioluminescent organisms. The oxyluciferin of bioluminescent dinoflagellates is not fluorescent, whereas its luciferin emits bright fluorescence with similar wavelength of the bioluminescence. What is the light emitter of dinoflagellate bioluminescence and what is the chemical process of the light emission like? These questions have not been answered by the limited experimental evidence so far. In this study, for the first time, the density functional calculation is employed to investigate the geometries and properties of luciferin and oxyluciferin of bioluminescent dinoflagellate. The calculated results agree with the experimental observations and indicate the luciferin or its analogue, rather than oxyluciferin, is the bioluminophore of dinoflagellate bioluminescence. A rough mechanism involving energy transfer is proposed for dinoflagellate bioluminescence.

  14. Fibre based cellular transfection.

    PubMed

    Tsampoula, X; Taguchi, K; Cizmár, T; Garces-Chavez, V; Ma, N; Mohanty, S; Mohanty, K; Gunn-Moore, F; Dholakia, K

    2008-10-13

    Optically assisted transfection is emerging as a powerful and versatile method for the delivery of foreign therapeutic agents to cells at will. In particular the use of ultrashort pulse lasers has proved an important route to transiently permeating the cell membrane through a multiphoton process. Though optical transfection has been gaining wider usage to date, all incarnations of this technique have employed free space light beams. In this paper we demonstrate the first system to use fibre delivery for the optical transfection of cells. We engineer a standard optical fibre to generate an axicon tip with an enhanced intensity of the remote output field that delivers ultrashort (~ 800 fs) pulses without requiring the fibre to be placed in very close proximity to the cell sample. A theoretical model is also developed in order to predict the light propagation from axicon tipped and bare fibres, in both air and water environments. The model proves to be in good agreement with the experimental findings and can be used to establish the optimum fibre parameters for successful cellular transfection. We readily obtain efficiencies of up to 57 % which are comparable with free space transfection. This advance paves the way for optical transfection of tissue samples and endoscopic embodiments of this technique.

  15. A single-cell bioluminescence imaging system for monitoring cellular gene expression in a plant body.

    PubMed

    Muranaka, Tomoaki; Kubota, Saya; Oyama, Tokitaka

    2013-12-01

    Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body.

  16. Stably transfected adherent cancer cell models with decreased expression of 5'-nucleotidase cN-II.

    PubMed

    Bricard, Gabriel; Cros-Perrial, Emeline; Machon, Christelle; Dumontet, Charles; Jordheim, Lars Petter

    2016-12-01

    The 5'-nucleotidase cN-II has been shown to be associated with the sensitivity to nucleoside analogues, the survival of cytarabine treated leukemia patients and to cell proliferation. Due to the lack of relevant cell models for solid tumors, we developed four cell lines with low cN-II expression and characterized them concerning their in vitro sensitivity to cancer drugs and their intracellular nucleotide pools. All four cell models had an important decrease of cN-II expression but did not show modified sensitivity, cell proliferation or nucleotide pools. Our cell models will be important for the study of the role of cN-II in human cancer cells.

  17. Marine Bioluminescence: Mechanisms and Evaluation

    DTIC Science & Technology

    1998-09-30

    study of bioluminescence in the S. California Bight using moored detectors, (3) continue study of luminescence in gelatinous zooplankton and marine snow...preparation were the principal efforts in the work on gelatinous zooplankton and marine snow. (4) Cytoskeletal investigations of Pyrocystis...potential adaptive significance of the wavelengths of light produced by gelatinous zooplankton . Bioluminescence spectra were measured from 100

  18. Graphene based gene transfection

    NASA Astrophysics Data System (ADS)

    Feng, Liangzhu; Zhang, Shuai; Liu, Zhuang

    2011-03-01

    Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI-10k polymer. The positively charged GO-PEI complexes are able to further bind with plasmid DNA (pDNA) for intracellular transfection of the enhanced green fluorescence protein (EGFP) gene in HeLa cells. While EGFP transfection with PEI-1.2k appears to be ineffective, high EGFP expression is observed using the corresponding GO-PEI-1.2k as the transfection agent. On the other hand, GO-PEI-10k shows similar EGFP transfection efficiency but lower toxicity compared with PEI-10k. Our results suggest graphene to be a novel gene delivery nano-vector with low cytotoxicity and high transfection efficiency, promising for future applications in non-viral based gene therapy.Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI

  19. Developing a Predictive Capability for Bioluminescence Signatures

    DTIC Science & Technology

    2010-01-01

    naval nighttime operations because the flow field associated with their motion stimulates naturally occurring plankton . In the littoral, the primary...sources of bioluminescence are dinoflagellates, common unicellular plankton that are also known to form red tides. Dinoflagellate bioluminescence is...bioluminescent “signatures” of some swimming fish are distinct enough to differentiate species; nocturnally foraging predators may use bioluminescent

  20. Developing a Predictive Capability for Bioluminescence Signatures

    DTIC Science & Technology

    2011-09-30

    naval nighttime operations because the flow field associated with their motion stimulates naturally occurring plankton . In the littoral, the primary...sources of bioluminescence are dinoflagellates, common unicellular plankton that are also known to form red tides. Dinoflagellate bioluminescence is...bioluminescent signatures of some swimming fish are distinct enough to differentiate species; nocturnally foraging predators may use bioluminescent

  1. Transfection using DEAE-dextran.

    PubMed

    Selden, R F

    2001-05-01

    Two protocols for DEAE-dextran transfection of cells are provided in this unit. The Basic Protocol describes a procedure used to transfect adherent cells and the first Alternate Protocol presents a method used to transfect suspension cells. If an increase in transfection efficiency is needed, cells can be treated with chloroquine as described in the second Alternate Protocol.

  2. [Inhibiting GDF-8 expression by retrovirus-based RNAi stably].

    PubMed

    Liu, Chaowu; Yang, Zhuo; Zhao, Bin; Liu, Changmei

    2008-02-01

    We cloned human U6 promoter from pAVU6 + 27 vector into pXSN to transcripe small RNA. Meanwhile, a shRNA targeting GDF-8 was cloned down-stream of the hU6 promoter to construct recombinant vector. Then the packing cell GP-293 was co-transfected the recombinant with pVSV-G to gernarate virus particle. Resistant C2C12 cell pools were screened using G418. Levels of mRNA and protein of GDF-8 were tested by Real-Time PCR and western blotting. Cell proliferation and cell cycle were analyzed using MTT and FACS. The expression of GDF-8 was dramatically decreased by the retrovirus-based system in C2C12 cells. Cells proliferated effectively after integrating the recombinant. The cells in G0/G1 phase decreased by 13.7%, while cells in S phase increased by 14.9%. In conclusion, the retrovirus-based RNAi could be used to stably silence GDF-8. It can be a powerful tool in curing muscle atrophy.

  3. Chemiluminescence and bioluminescence microbe detection

    NASA Technical Reports Server (NTRS)

    Taylor, R. E.; Chappelle, E.; Picciolo, G. L.; Jeffers, E. L.; Thomas, R. R.

    1978-01-01

    Automated biosensors for online use with NASA Water Monitoring System employs bioluminescence and chemiluminescence techniques to rapidly measure microbe contamination of water samples. System eliminates standard laboratory procedures requiring time duration of 24 hours or longer.

  4. Bioluminescence Potential Modeling and Forecasting

    DTIC Science & Technology

    2013-05-22

    bioluminescence in the wakes of ships, breaking waves, around the bodies of rapidly moving fish and mammals , and from simple agitation of the water with one’s hand...history of brilliant displays of bioluminescence in the wakes of ships, breaking waves, around the bodies of rapidly moving fish and mammals , and from...during the earlier stages of upwelling development. Later, the observed deep offshore BL potential maximum disappeared and became a shallower and much

  5. Temperature-modulated bioluminescence tomography

    NASA Astrophysics Data System (ADS)

    Wang, Ge; Shen, Haiou; Cong, Wenxiang; Zhao, Shan; Wei, Guo Wei

    2006-08-01

    It was recently reported that bioluminescent spectra can be significantly affected by temperature, which we recognize as a major opportunity to overcome the inherent illposedness of bioluminescence tomography (BLT). In this paper, we propose temperature-modulated bioluminescence tomography (TBT) to utilize the temperature dependence of bioluminescence for superior BLT performance. Specifically, we employ a focused ultrasound array to heat small volumes of interest (VOI) one at a time, and induce a detectable change in the optical signal on the body surface of a mouse. Based on this type of information, the BLT reconstruction can be stabilized and improved. Our numerical experiments clearly demonstrate the merits of our TBT with either noise-free or noisy datasets. Also, this idea is applicable in 2D bioluminescence imaging and computational optical biopsy (COB). We believe that our approach and technology represents a major step forward in the field of BLT, and has an important and immediate applicability in bioluminescence imaging of small animals in general.

  6. Noninvasive bioluminescence imaging of dengue virus infection in the brain of A129 mice.

    PubMed

    Li, Xiao-Feng; Deng, Yong-Qiang; Zhao, Hui; Ye, Qing; Wang, Hong-Jiang; Li, Shi-Hua; Zhu, Shun-Ya; Shi, Pei-Yong; Qin, E-De; Zhang, Bo; Qin, Cheng-Feng

    2013-05-01

    Dengue virus (DENV) infection is one of the most important public health threats globally; however, no vaccines or effective antivirals are currently available. The bioluminescence imaging technique has emerged as a powerful tool for studies on viral pathogenesis in vitro and in vivo. In this study, using a recombinant DENV that stably expressed Renilla luciferase (Rluc-DENV), we used bioluminescence for imaging of DENV infection in the brain of A129 mice that lacked type I interferon receptors. Upon intracranial inoculation with Rluc-DENV, A129 mice developed typical neurological symptoms and rapidly succumbed to viral infection. Real-time bioluminescence intensity analysis revealed the replication kinetics of Rluc-DENV in the brain of A129 mice. Linear regression analyses showed a good correlation between photon flux and viral titers (R(2) = 0.9923). Finally, the bioluminescence model was validated using a known mouse monoclonal antibody, 2A10G6, and the therapeutic effects of this neutralizing antibody were readily monitored by live imaging in the same animal. The noninvasive bioluminescence imaging of DENV infection as described here shows distinct advantages over traditional animal models and provides a powerful tool for potential antiviral or vaccine assays against DENV infection in vivo.

  7. Developing a Predictive Capability for Bioluminescence Signatures

    DTIC Science & Technology

    2009-01-01

    to flow stimulation of naturally occurring plankton. In the littoral, the primary sources of bioluminescence are dinoflagellates, common unicellular ...littoral, the primary sources of bioluminescence are dinoflagellates, common unicellular plankton that exhibit a wide range of abundance. Dinoflagellate

  8. Engineering Bioluminescent Proteins: Expanding their Analytical Potential

    PubMed Central

    Rowe, Laura; Dikici, Emre; Daunert, Sylvia

    2009-01-01

    Synopsis Bioluminescence has been observed in nature since the dawn of time, but now, scientists are harnessing it for analytical applications. Laura Rowe, Emre Dikici, and Sylvia Daunert of the University of Kentucky describe the origins of bioluminescent proteins and explore their uses in the modern chemistry laboratory. The cover features spectra of bioluminescent light superimposed on an image of jellyfish, which are a common source of bioluminescent proteins. Images courtesy of Emre Dikici and Shutterstock. PMID:19725502

  9. Plasma-mediated transfection of RPE

    NASA Astrophysics Data System (ADS)

    Palanker, D.; Chalberg, T.; Vankov, A.; Huie, P.; Molnar, F. E.; Butterwick, A.; Calos, M.; Marmor, M.; Blumenkranz, M. S.

    2006-02-01

    A major obstacle in applying gene therapy to clinical practice is the lack of efficient and safe gene delivery techniques. Viral delivery has encountered a number of serious problems including immunological reactions and malignancy. Non-viral delivery methods (liposomes, sonoporation and electroporation) have either low efficiency in-vivo or produce severe collateral damage to ocular tissues. We discovered that tensile stress greatly increases the susceptibility of cellular membranes to electroporation. For synchronous application of electric field and mechanical stress, both are generated by the electric discharge itself. A pressure wave is produced by rapid vaporization of the medium. To prevent termination of electric current by the vapor cavity it is ionized thus restoring its electric conductivity. For in-vivo experiments with rabbits a plasmid DNA was injected into the subretinal space, and RPE was treated trans-sclerally with an array of microelectodes placed outside the eye. Application of 250-300V and 100-200 μs biphasic pulses via a microelectrode array resulted in efficient transfection of RPE without visible damage to the retina. Gene expression was quantified and monitored using bioluminescence (luciferase) and fluorescence (GFP) imaging. Transfection efficiency of RPE with this new technique exceeded that of standard electroporation by a factor 10,000. Safe and effective non-viral DNA delivery to the mammalian retina may help to materialize the enormous potential of the ocular gene therapy. Future experiments will focus on continued characterization of the safety and efficacy of this method and evaluation of long-term transgene expression in the presence of phiC31 integrase.

  10. Homogeneous, bioluminescent proteasome assays.

    PubMed

    O'Brien, Martha A; Moravec, Richard A; Riss, Terry L; Bulleit, Robert F

    2015-01-01

    Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

  11. Transfection using DEAE-dextran.

    PubMed

    Gulick, T

    2001-05-01

    Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

  12. Transfection using DEAE-dextran.

    PubMed

    Gulick, Tod

    2003-08-01

    Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

  13. Transfection using DEAE-dextran.

    PubMed

    Gulick, T

    2001-05-01

    Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The Basic Protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

  14. Bioluminescent Reaction by Immobilized Luciferase

    NASA Astrophysics Data System (ADS)

    Tanaka, Ryuta; Takahama, Eriko; Iinuma, Masataka; Ikeda, Takeshi; Kadoya, Yutaka; Kuroda, Akio

    We have investigated an effect of immobilization of luciferase molecules at the optical fiber end on a bioluminescent reaction. The time dependence of measured count rates of emitted photons has been analyzed by fitting with numerical solution of differential equations including the effect of the product-inhibitor and the deactivation of the luciferase. Through the analysis, we have successfully extracted kinetic constants such as, reaction rate, number of active luciferase molecules, etc. Ratio of active molecules to total luciferase molecules in immobilization was one order of magnitude lower than that in solution. The reaction rate of the bioluminescent process was also different from the one of free luciferase in solution.

  15. Transfection of Platyhelminthes

    PubMed Central

    Moguel, Bárbara; Bobes, Raúl J.; Carrero, Julio C.; Laclette, Juan P.

    2015-01-01

    Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species. PMID:26090388

  16. Transfection of Platyhelminthes.

    PubMed

    Moguel, Bárbara; Bobes, Raúl J; Carrero, Julio C; Laclette, Juan P

    2015-01-01

    Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species.

  17. Biofilm Ecology of Bioluminescent Bacteria

    DTIC Science & Technology

    1992-08-10

    F.V. Lamberti , Z. Policova, W. Zingg, C.J. van Oss, and A.W. Neumann. 1983. Surface thermodynamics of bacterial adhesion. Appl. Environ. Microbiol. 46...of bacterial adhesion in a shear gradient with bioluminescence by a Pseudomonas fluorescens (lux) strain . J. Microbiol. Meth. 15:53-60. Mittelman, M.W

  18. Migration of Adipose-derived Mesenchymal Stem Cells Stably Expressing Chondroitinase ABC In vitro

    PubMed Central

    Wu, Jian-Huang; Li, Miao; Liang, Yan; Lu, Tao; Duan, Chun-Yue

    2016-01-01

    Background: Several studies have revealed that adipose-derived mesenchymal stem cells (ADSCs) can be used as seed cells for the treatment of spinal cord injury (SCI). Chondroitinase ABC (ChABC) decomposes chondroitin sulfate proteoglycans in the glial scar that forms following SCI, allowing stem cells to penetrate through the scar and promote recovery of nerve function. This study aimed to establish ADSCs that stably express ChABC (ChABC-ADSCs) and evaluate the migratory capability of ChABC-ADSCs in vitro. Methods: ADSCs were obtained from Sprague-Dawley rats using secondary collagenase digestion. Their phenotypes were characterized using flow cytometry detection of cell surface antigens and their stem cell properties were confirmed by induction of differentiation. After successful culture, ADSCs were transfected with lentiviral vectors and ChABC-ADSCs were obtained. Proliferation curves of ChABC-ADSCs were determined using the Cell Counting Kit-8 method, ChABC expression was verified using Western blotting, and the migration of ChABC-ADSCs was analyzed using the transwell assay. Results: Secondary collagenase digestion increased the isolation efficiency of primary ADSCs. Following transfection using lentiviral vectors, the proliferation of ChABC-ADSCs was reduced in comparison with control ADSCs at 48 h (P < 0.05). And the level of ChABC expression in the ChABC-ADSC group was significantly higher than that of the ADSC group (P < 0.05). Moreover, ChABC-ADSC migration in matrigel was significantly enhanced in comparison with the control (P < 0.05). Conclusions: Secondary collagenase digestion can be used to effectively isolate ADSCs. ChABC-ADSCs constructed using lentiviral vector transfection stably express ChABC, and ChABC expression significantly enhances the migratory capacity of ADSCs. PMID:27364797

  19. Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

    PubMed Central

    Xu, Tingting; Close, Dan M.; Webb, James D.; Ripp, Steven A.; Sayler, Gary S.

    2013-01-01

    Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion. PMID:24193545

  20. Combining fluorescence and bioluminescence microscopy.

    PubMed

    Goda, Kazuhito; Hatta-Ohashi, Yoko; Akiyoshi, Ryutaro; Sugiyama, Takashi; Sakai, Ikuko; Takahashi, Takeo; Suzuki, Hirobumi

    2015-08-01

    Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells.

  1. Bioluminescence imaging in live cells and animals.

    PubMed

    Tung, Jack K; Berglund, Ken; Gutekunst, Claire-Anne; Hochgeschwender, Ute; Gross, Robert E

    2016-04-01

    The use of bioluminescent reporters in neuroscience research continues to grow at a rapid pace as their applications and unique advantages over conventional fluorescent reporters become more appreciated. Here, we describe practical methods and principles for detecting and imaging bioluminescence from live cells and animals. We systematically tested various components of our conventional fluorescence microscope to optimize it for long-term bioluminescence imaging. High-resolution bioluminescence images from live neurons were obtained with our microscope setup, which could be continuously captured for several hours with no signs of phototoxicity. Bioluminescence from the mouse brain was also imaged noninvasively through the intact skull with a conventional luminescence imager. These methods demonstrate how bioluminescence can be routinely detected and measured from live cells and animals in a cost-effective way with common reagents and equipment.

  2. Bioluminescence assay for cell viability.

    PubMed

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N

    2015-06-01

    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  3. Uniqueness theorems in bioluminescence tomography.

    PubMed

    Wang, Ge; Li, Yi; Jiang, Ming

    2004-08-01

    Motivated by bioluminescent imaging needs for studies on gene therapy and other applications in the mouse models, a bioluminescence tomography (BLT) system is being developed in the University of Iowa. While the forward imaging model is described by the well-known diffusion equation, the inverse problem is to recover an internal bioluminescent source distribution subject to Cauchy data. Our primary goal in this paper is to establish the solution uniqueness for BLT under practical constraints despite the ill-posedness of the inverse problem in the general case. After a review on the inverse source literature, we demonstrate that in the general case the BLT solution is not unique by constructing the set of all the solutions to this inverse problem. Then, we show the uniqueness of the solution in the case of impulse sources. Finally, we present our main theorem that solid/hollow ball sources can be uniquely determined up to nonradiating sources. For better readability, the exact conditions for and rigorous proofs of the theorems are given in the Appendices. Further research directions are also discussed.

  4. Microbiological assay using bioluminescent organism

    SciTech Connect

    Stiffey, A.V.

    1987-12-21

    This invention relates to testing processes for toxicity involving microorganisms and, more particularly, to testing processes for toxicity involving bioluminescent organisms. The present known method of testing oil-well drilling fluids for toxicity employs the mysid shrimp (Mysidopsis bahia) as the assay organism. The shrimp are difficult to raise and handle as laboratory assay organisms. This method is labor-intensive, because it requires a assay time of about 96 hours. Summary of the Invention: A microbiological assay in which the assay organism is the dinoflagellate, Pyrocystis lunula. A sample of a substance to be assayed is added to known numbers of the bioluminescent dinoflagellate and the mixture is agitated to subject the organisms to a shear stress causing them to emit light. The amount of light emitted is measured and compared with the amount of light emitted by a known non-toxic control mixture to determine if there is diminution or non-diminution of light emitted by the sample under test which is an indication of the presence or absence of toxicity, respectively. Accordingly, an object of the present invention is the provision of an improved method of testing substances for toxicity. A further object of the invention is the provision of an improved method of testing oil-well drilling fluids for toxicity using bioluminescent dinoflagellate (Pyrocystis lunula).

  5. Murine Bioluminescent Hepatic Tumour Model

    PubMed Central

    Rajendran, Simon; Salwa, Slawomir; Gao, Xuefeng; Tabirca, Sabin; O'Hanlon, Deirdre; O'Sullivan, Gerald C.; Tangney, Mark

    2010-01-01

    This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. PMID:20689502

  6. Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents.

    PubMed

    Chettab, Kamel; Roux, Stéphanie; Mathé, Doriane; Cros-Perrial, Emeline; Lafond, Maxime; Lafon, Cyril; Dumontet, Charles; Mestas, Jean-Louis

    2015-01-01

    Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. In this study, we developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. The regulation system incorporated in the device allowed a real-time control of the cavitation level during sonoporation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (pEGFP-C1) in adherent and non-adherent cell lines. The transfection efficiency of the device was compared to those observed with lipofection and nucleofection methods. In both adherent and non-adherent cell lines, the sonoporation device allowed high rate of transfection of pEGFP-C1 (40-80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. The transfection efficiency and toxicity of sonoporation on the non-adherent cell lines Jurkat and K562 were similar to those of nucleofection, while these two cell lines were resistant to transfection by lipofection. Moreover, sonoporation was used to produce three stably transfected human lymphoma and leukemia lines. Significant transfection efficiency was also observed in two fresh samples of human acute myeloid leukemia cells. In conclusion, we developed a user-friendly and cost-effective ultrasound device, well adapted for routine in vitro high-yield transfection experiments and which does not require the use of any transfection reagent or gas micro-bubbles.

  7. Assessing laser-tissue damage with bioluminescent imaging.

    PubMed

    Wilmink, Gerald J; Opalenik, Susan R; Beckham, Joshua T; Davidson, Jeffrey M; Jansen, E Duco

    2006-01-01

    Effective medical laser procedures are achieved by selecting laser parameters that minimize undesirable tissue damage. Traditionally, human subjects, animal models, and monolayer cell cultures have been used to study wound healing, tissue damage, and cellular effects of laser radiation. Each of these models has significant limitations, and consequently, a novel skin model is needed. To this end, a highly reproducible human skin model that enables noninvasive and longitudinal studies of gene expression was sought. In this study, we present an organotypic raft model (engineered skin) used in combination with bioluminescent imaging (BLI) techniques. The efficacy of the raft model was validated and characterized by investigating the role of heat shock protein 70 (hsp70) as a sensitive marker of thermal damage. The raft model consists of human cells incorporated into an extracellular matrix. The raft cultures were transfected with an adenovirus containing a murine hsp70 promoter driving transcription of luciferase. The model enables quantitative analysis of spatiotemporal expression of proteins using BLI. Thermal stress was induced on the raft cultures by means of a constant temperature water bath or with a carbon dioxide (CO2) laser (lambda=10.6 microm, 0.679 to 2.262 Wcm2, cw, unfocused Gaussian beam, omegaL=4.5 mm, 1 min exposure). The bioluminescence was monitored noninvasively with an IVIS 100 Bioluminescent Imaging System. BLI indicated that peak hsp70 expression occurs 4 to 12 h after exposure to thermal stress. A minimum irradiance of 0.679 Wcm2 activated the hsp70 response, and a higher irradiance of 2.262 Wcm2 was associated with a severe reduction in hsp70 response due to tissue ablation. Reverse transcription polymerase chain reaction demonstrated that hsp70 mRNA levels increased with prolonged heating exposures. Enzyme-linked immunosorbent protein assays confirmed that luciferase was an accurate surrogate for hsp70 intracellular protein levels. Hematoxylin

  8. Assessing laser-tissue damage with bioluminescent imaging

    NASA Astrophysics Data System (ADS)

    Wilmink, Gerald J.; Opalenik, Susan R.; Beckham, Josh T.; Davidson, Jeffrey M.; Jansen, Eric D.

    2006-07-01

    Effective medical laser procedures are achieved by selecting laser parameters that minimize undesirable tissue damage. Traditionally, human subjects, animal models, and monolayer cell cultures have been used to study wound healing, tissue damage, and cellular effects of laser radiation. Each of these models has significant limitations, and consequently, a novel skin model is needed. To this end, a highly reproducible human skin model that enables noninvasive and longitudinal studies of gene expression was sought. In this study, we present an organotypic raft model (engineered skin) used in combination with bioluminescent imaging (BLI) techniques. The efficacy of the raft model was validated and characterized by investigating the role of heat shock protein 70 (hsp70) as a sensitive marker of thermal damage. The raft model consists of human cells incorporated into an extracellular matrix. The raft cultures were transfected with an adenovirus containing a murine hsp70 promoter driving transcription of luciferase. The model enables quantitative analysis of spatiotemporal expression of proteins using BLI. Thermal stress was induced on the raft cultures by means of a constant temperature water bath or with a carbon dioxide (CO2) laser (λ=10.6 µm, 0.679 to 2.262 W/cm2, cw, unfocused Gaussian beam, ωL=4.5 mm, 1 min exposure). The bioluminescence was monitored noninvasively with an IVIS 100 Bioluminescent Imaging System. BLI indicated that peak hsp70 expression occurs 4 to 12 h after exposure to thermal stress. A minimum irradiance of 0.679 W/cm2 activated the hsp70 response, and a higher irradiance of 2.262 W/cm2 was associated with a severe reduction in hsp70 response due to tissue ablation. Reverse transcription polymerase chain reaction demonstrated that hsp70 mRNA levels increased with prolonged heating exposures. Enzyme-linked immunosorbent protein assays confirmed that luciferase was an accurate surrogate for hsp70 intracellular protein levels. Hematoxylin and

  9. Stable transfection into rat bone marrow mesenchymal stem cells by lentivirus-mediated NT-3.

    PubMed

    Gong, Yu; Wang, Hongfei; Xia, Haijun

    2015-01-01

    Transplantation of bone marrow mesenchymal stem cells (BMSCs) is the most promising therapeutic strategy in the treatment of spinal cord injuries. BMSCs have a wide variety of sources and are characterized by being exempt from immune rejection, marked secretory functions and neuronal plasticity during differentiation. The lentiviral vector, namely PLV.Ex3d.P/neo-EF1A-NT3-internal ribosome entry site-enhanced green fluorescent protein, was constructed and subsequently transfected into Sprague Dawley (SD) rat BMSCs. The gene and protein expression levels of the nucleic acid neurotrophin-3 (NT-3) were then detected. The results demonstrated that the constructed NT-3 gene lentiviral expression vector matched the expected design and that the NT-3 gene was transfected into the BMSCs via the lentivirus‑mediated method at a transfection efficiency of 60‑70%. NT-3 gene expression was detected within the stably transfected positive cells at the nucleic acid and protein levels. The cell morphology and biological activity of BMSCs did not alter significantly following transfection with NT-3. NT-3-transfected SD BMSCs were successfully constructed and served as effective vector seed cells with stable expression. These results can be used as a reference for subsequent studies on the transplantation therapy of rat spinal cord injuries using lentivirus-mediated NT-3-transfected SD BMSCs.

  10. Quick preparation of nanoluciferase-based tracers for novel bioluminescent receptor-binding assays of protein hormones: Using erythropoietin as a model.

    PubMed

    Song, Ge; Wu, Qing-Ping; Xu, Ting; Liu, Ya-Li; Xu, Zeng-Guang; Zhang, Shi-Fu; Guo, Zhan-Yun

    2015-12-01

    Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the so far brightest bioluminescence. In recent studies, we developed NanoLuc as an ultrasensitive probe for novel bioluminescent receptor-binding assays of some protein/peptide hormones. In the present study, we proposed a simple method for quick preparation of the NanoLuc-based protein tracers using erythropoietin (Epo) as a model. Epo is a glycosylated cytokine that promotes erythropoiesis by binding and activating the cell membrane receptor EpoR. For quick preparation of a bioluminescent Epo tracer, an Epo-Luc fusion protein carrying a NanoLuc-6 × His-tag at the C-terminus was secretorily overexpressed in transiently transfected human embryonic kidney (HEK) 293 T cells. The Epo-Luc fusion protein retained high-binding affinities with EpoR either overexpressed in HEK293T cells or endogenously expressed in mouse erythroleukemia cells, representing a novel ultrasensitive bioluminescent tracer for non-radioactive receptor-binding assays. Sufficient Epo-Luc tracer for thousands of assays could be quickly obtained within 2 days through simple transient transfection. Thus, our present work provided a simple method for quick preparation of novel NanoLuc-based bioluminescent tracers for Epo and some other protein hormones to facilitate their ligand-receptor interaction studies.

  11. Bioluminescent bacterial imaging in vivo.

    PubMed

    Baban, Chwanrow K; Cronin, Michelle; Akin, Ali R; O'Brien, Anne; Gao, Xuefeng; Tabirca, Sabin; Francis, Kevin P; Tangney, Mark

    2012-11-04

    This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

  12. The Chemical Basis of Fungal Bioluminescence.

    PubMed

    Purtov, Konstantin V; Petushkov, Valentin N; Baranov, Mikhail S; Mineev, Konstantin S; Rodionova, Natalja S; Kaskova, Zinaida M; Tsarkova, Aleksandra S; Petunin, Alexei I; Bondar, Vladimir S; Rodicheva, Emma K; Medvedeva, Svetlana E; Oba, Yuichi; Oba, Yumiko; Arseniev, Alexander S; Lukyanov, Sergey; Gitelson, Josef I; Yampolsky, Ilia V

    2015-07-06

    Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

  13. Repeated and Widespread Evolution of Bioluminescence in Marine Fishes

    PubMed Central

    Davis, Matthew P.; Sparks, John S.; Smith, W. Leo

    2016-01-01

    Bioluminescence is primarily a marine phenomenon with 80% of metazoan bioluminescent genera occurring in the world’s oceans. Here we show that bioluminescence has evolved repeatedly and is phylogenetically widespread across ray-finned fishes. We recover 27 independent evolutionary events of bioluminescence, all among marine fish lineages. This finding indicates that bioluminescence has evolved many more times than previously hypothesized across fishes and the tree of life. Our exploration of the macroevolutionary patterns of bioluminescent lineages indicates that the present day diversity of some inshore and deep-sea bioluminescent fish lineages that use bioluminescence for communication, feeding, and reproduction exhibit exceptional species richness given clade age. We show that exceptional species richness occurs particularly in deep-sea fishes with intrinsic bioluminescent systems and both shallow water and deep-sea lineages with luminescent systems used for communication. PMID:27276229

  14. Repeated and Widespread Evolution of Bioluminescence in Marine Fishes.

    PubMed

    Davis, Matthew P; Sparks, John S; Smith, W Leo

    2016-01-01

    Bioluminescence is primarily a marine phenomenon with 80% of metazoan bioluminescent genera occurring in the world's oceans. Here we show that bioluminescence has evolved repeatedly and is phylogenetically widespread across ray-finned fishes. We recover 27 independent evolutionary events of bioluminescence, all among marine fish lineages. This finding indicates that bioluminescence has evolved many more times than previously hypothesized across fishes and the tree of life. Our exploration of the macroevolutionary patterns of bioluminescent lineages indicates that the present day diversity of some inshore and deep-sea bioluminescent fish lineages that use bioluminescence for communication, feeding, and reproduction exhibit exceptional species richness given clade age. We show that exceptional species richness occurs particularly in deep-sea fishes with intrinsic bioluminescent systems and both shallow water and deep-sea lineages with luminescent systems used for communication.

  15. Transfected cell lines as tools for high throughput screening: a call for standards.

    PubMed

    Pagliaro, L; Praestegaard, M

    2001-06-01

    During 1999, Journal of Biomolecular Screening presented a series of Point-Counterpoint articles that addressed a question posed by editor Bill Janzen: "What is the future of HTS?" These articles discussed many of the global issues involved in HTS, such as target identification and library size, as well as the scientific and technical challenges facing the field. In this perspective we address a related, but very focused, issue that is increasingly important for many of us in the HTS community: the use of stably transfected cell lines as an integral part of screening strategies. Transfected cell lines provide powerful tools for assay design, but at the same time they introduce complex variables into the screening system. Although it is difficult to develop precise definitions and standards for biologicals such as cell lines, we propose that the development of guidelines for the nomenclature and use of transfected cell lines is essential for their use in HTS.

  16. Analytical Applications of Bioluminescence and Chemiluminescence

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W. (Editor); Picciolo, G. L. (Editor)

    1975-01-01

    Bioluminescence and chemiluminescence studies were used to measure the amount of adenosine triphosphate and therefore the amount of energy available. Firefly luciferase - luciferin enzyme system was emphasized. Photometer designs are also considered.

  17. Ecology of colors of firefly bioluminescence

    SciTech Connect

    Lall, A.B.; Seliger, H.H.; Biggley, W.H.; Lloyd, J.E.

    1980-10-31

    Dark-active North American fireflies emit green bioluminescence and dusk-active species emit yellow, in general. Yellow light and yellow visual spectral sensitivity may be adaptations to increase the signal-to-noise (that is, foliage-reflected ambient light) ratio for sexual signaling during twilight. The peaks of the electroretinogram visual spectral sensitivities of four species tested, two dark- and two dusk-active, correspond with the peak of their bioluminescent emissions.

  18. Circadian control sheds light on fungal bioluminescence.

    PubMed

    Oliveira, Anderson G; Stevani, Cassius V; Waldenmaier, Hans E; Viviani, Vadim; Emerson, Jillian M; Loros, Jennifer J; Dunlap, Jay C

    2015-03-30

    Bioluminescence, the creation and emission of light by organisms, affords insight into the lives of organisms doing it. Luminous living things are widespread and access diverse mechanisms to generate and control luminescence [1-5]. Among the least studied bioluminescent organisms are phylogenetically rare fungi-only 71 species, all within the ∼ 9,000 fungi of the temperate and tropical Agaricales order-are reported from among ∼ 100,000 described fungal species [6, 7]. All require oxygen [8] and energy (NADH or NADPH) for bioluminescence and are reported to emit green light (λmax 530 nm) continuously, implying a metabolic function for bioluminescence, perhaps as a byproduct of oxidative metabolism in lignin degradation. Here, however, we report that bioluminescence from the mycelium of Neonothopanus gardneri is controlled by a temperature-compensated circadian clock, the result of cycles in content/activity of the luciferase, reductase, and luciferin that comprise the luminescent system. Because regulation implies an adaptive function for bioluminescence, a controversial question for more than two millennia [8-15], we examined interactions between luminescent fungi and insects [16]. Prosthetic acrylic resin "mushrooms," internally illuminated by a green LED emitting light similar to the bioluminescence, attract staphilinid rove beetles (coleopterans), as well as hemipterans (true bugs), dipterans (flies), and hymenopterans (wasps and ants), at numbers far greater than dark control traps. Thus, circadian control may optimize energy use for when bioluminescence is most visible, attracting insects that can in turn help in spore dispersal, thereby benefitting fungi growing under the forest canopy, where wind flow is greatly reduced.

  19. Circadian Control Sheds Light on Fungal Bioluminescence

    PubMed Central

    Oliveira, Anderson G.; Stevani, Cassius V.; Waldenmaier, Hans E.; Viviani, Vadim; Emerson, Jillian M.; Loros, Jennifer J.; Dunlap, Jay C.

    2015-01-01

    Summary Bioluminescence, the creation and emission of light by organisms, affords insight into the lives of organisms doing it. Luminous living things are widespread and access diverse mechanisms to generate and control luminescence [1-5]. Among the least studied bioluminescent organisms are phylogenetically rare fungi – only 71 species, all within the ~9000 fungi of the temperate and tropical Agaricales Order - are reported from among ~100,000 described fungal species [6,7]. All require oxygen [8] and energy (NADH or NADPH) for bioluminescence, and are reported to emit green light (λmax 530 nm) continuously, implying a metabolic function for bioluminescence, perhaps as a by-product of oxidative metabolism in lignin degradation. Here, however, we report that bioluminescence from the mycelium of Neonothopanus gardneri is controlled by a temperature compensated circadian clock, the result of cycles in content/activity of the luciferase, reductase, and the luciferin that comprise the luminescent system. Because regulation implies an adaptive function for bioluminescence, a controversial question for more than two millenia [8-15], we examined interactions between luminescent fungi and insects [16]. Prosthetic acrylic resin “mushrooms”, internally illuminated by a green LED emitting light similar to the bioluminescence, attract staphilinid rove beetles (coleopterans) as well as hemipterans (true bugs), dipterans (flies), and hymenopterans (wasps and ants) at numbers far greater than dark control traps. Thus, circadian control may optimize energy use for when bioluminescence is most visible, attracting insects that can in turn help in spore dispersal, thereby benefitting fungi growing under the forest canopy where wind flow is greatly reduced. PMID:25802150

  20. Bioluminescence in Dinoflagellates: Evidence that the Adaptive Value of Bioluminescence in Dinoflagellates is Concentration Dependent.

    PubMed

    Hanley, Karen A; Widder, Edith A

    2017-03-01

    Three major hypotheses have been proposed to explain why dinoflagellate bioluminescence deters copepod grazing: startle response, aposematic warning, and burglar alarm. These hypotheses propose dinoflagellate bioluminescence (A) startles predatory copepods, (B) warns potential predators of toxicity, and (C) draws the attention of higher order visual predators to the copepod's location. While the burglar alarm is the most commonly accepted hypothesis, it requires a high concentration of bioluminescent dinoflagellates to be effective, meaning the bioluminescence selective advantage at lower, more commonly observed, dinoflagellate concentrations may result from another function (e.g. startle response or aposematic warning). Therefore, a series of experiments was conducted to evaluate copepod grazing (Acartia tonsa) on bioluminescent dinoflagellates (during bioluminescent and nonbioluminescent phases, corresponding to night and day, respectively) at different concentrations (10, 1000, and 3000 cells mL(-1) ), on toxic (Pyrodinium bahamense var. bahamense) and nontoxic (Lingulodinium polyedrum) bioluminescent dinoflagellates, and in the presence of nonluminescent diatoms (Thalassiosira eccentrica). Changes in copepod ingestion rates, clearance rates, and feeding preferences as a result of these experimental factors, particularly during the mixed trails with nonluminescent diatoms, indicate there is a concentration threshold at which the burglar alarm becomes effective and below which dinoflagellate bioluminescence functions as an aposematic warning.

  1. Calcium Phosphate Transfection of Primary Hippocampal Neurons

    PubMed Central

    DiBona, Victoria L.; Wu, Qian; Zhang, Huaye

    2013-01-01

    Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells. PMID:24300106

  2. Bidirectional cytoplasmic incompatibility induced by cross-order transfection of Wolbachia: implications for control of the host population.

    PubMed

    Zhong, Yong; Li, Zheng-Xi

    2014-10-01

    Wolbachia are widespread endosymbionts in arthropods and some nematodes. This genus of bacteria is known to manipulate host reproduction by inducing cytoplasmic incompatibility (CI). This important phenotype is implicated in the control of host populations since Wolbachia can suppress host populations through the induction of CI in a way similar to the sterile insect technique. Here, we identified a candidate CI-inducing Wolbachia strain from the parasitic wasp Scleroderma guani (wSguBJ) by sequencing and phylogenetic analysis. This Wolbachia strain was then isolated, purified, and artificially transfected into the new whitefly host Bemisia tabaci through nymphal microinjection. Infection frequency monitoring by molecular detection showed that 60-80 % of the offspring from transfected whitefly populations was infected with wSguBJ six generations after the transfer. Laboratory rearing experiments indicated that the artificial transfection caused no significant difference in the numbers of offspring between the transfected and naturally infected populations and had no significant detrimental effects on the development of transfected males, although the development of transfected females was delayed. Reciprocal crossings revealed that bidirectional CI was induced between the transfected and naturally infected whiteflies. These data indicated that the cross-order transfer of the heterologous Wolbachia strain by nymphal microinjection was successful. Mass release of the transfected males that could stably carry the heterologous Wolbachia without significant compromise of fecundity/development may provide an alternative approach to control of host populations.

  3. Molecular genetic transfection of the coccidian parasite Sarcocystis neurona.

    PubMed

    Gaji, Rajshekhar Y; Zhang, Deqing; Breathnach, Cormac C; Vaishnava, Shipra; Striepen, Boris; Howe, Daniel K

    2006-11-01

    Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5' flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules beta-galactosidase (beta-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express beta-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.

  4. Discovery of New Substrates for LuxAB Bacterial Bioluminescence.

    PubMed

    Jiang, Tianyu; Wang, Weishan; Wu, Xingkang; Wu, Wenxiao; Bai, Haixiu; Ma, Zhao; Shen, Yuemao; Yang, Keqian; Li, Minyong

    2016-08-01

    In this article, four novel substrates with long halftime have been designed and synthesized successfully for luxAB bacterial bioluminescence. After in vitro and in vivo biological evaluation, these molecules can emit obvious bioluminescence emission with known bacterial luciferase, thus indicating a new promising approach to developing the bacterial bioluminescent system.

  5. Bioluminescence patterns among North American Armillaria species.

    PubMed

    Mihail, Jeanne D

    2015-06-01

    Bioluminescence is widely recognized among white-spored species of Basidiomycota. Most reports of fungal bioluminescence are based upon visual light perception. When instruments such as photomultipliers have been used to measure fungal luminescence, more taxa have been discovered to produce light, albeit at a range of magnitudes. The present studies were undertaken to determine the prevalence of bioluminescence among North American Armillaria species. Consistent, constitutive bioluminescence was detected for the first time for mycelia of Armillaria calvescens, Armillaria cepistipes, Armillaria gemina, Armillaria nabsnona, and Armillaria sinapina and confirmed for mycelia of Armillaria gallica, Armillaria mellea, Armillaria ostoyae, and Armillaria tabescens. Emission spectra of mycelia representing all species had maximum intensity in the range 515-525 nm confirming that emitted light was the result of bioluminescence rather than chemiluminescence. Time series analysis of 1000 consecutive luminescence measurements revealed a highly significant departure from random variation. Mycelial luminescence of eight species exhibited significant, stable shifts in magnitude in response to a series of mechanical disturbance treatments, providing one mechanism for generating observed luminescence variation.

  6. Immobilized Bioluminescent Reagents in Flow Injection Analysis.

    NASA Astrophysics Data System (ADS)

    Nabi, Abdul

    Available from UMI in association with The British Library. Bioluminescent reactions exhibits two important characteristics from an analytical viewpoint; they are selective and highly sensitive. Furthermore, bioluminescent emissions are easily measured with a simple flow-through detector based on a photomultiplier tube and the rapid and reproducible mixing of sample and expensive reagent is best achieved by a flow injection manifold. The two most important bioluminescent systems are the enzyme (luciferase)/substrate (luciferin) combinations extracted from fireflies (Photinus pyralis) and marine bacteria (Virio harveyi) which requires ATP and NAD(P)H respectively as cofactors. Reactions that generate or consume these cofactors can also be coupled to the bioluminescent reaction to provide assays for a wide range of clinically important species. A flow injection manifold for the study of bioluminescent reactions is described, as are procedures for the extraction, purification and immobilization of firefly and bacterial luciferase and oxidoreductase. Results are presented for the determination of ATP using firefly system and the determination of other enzymes and substrates participating in ATP-converting reactions e.g. creatine kinase, ATP-sulphurylase, pyruvate kinase, creatine phosphate, pyrophosphate and phophoenolypyruvate. Similarly results are presented for the determination of NAD(P)H, FMN, FMNH_2 and several dehydrogenases which produce NAD(P)H and their substrates, e.g. alcohol, L-lactate, L-malate, L-glutamate, Glucose-6-phosphate and primary bile acid.

  7. Fluorescent and Bioluminescent Reporter Myxoviruses

    PubMed Central

    Rostad, Christina A.; Currier, Michael C.; Moore, Martin L.

    2016-01-01

    The advent of virus reverse genetics has enabled the incorporation of genetically encoded reporter proteins into replication-competent viruses. These reporters include fluorescent proteins which have intrinsic chromophores that absorb light and re-emit it at lower wavelengths, and bioluminescent proteins which are luciferase enzymes that react with substrates to produce visible light. The incorporation of these reporters into replication-competent viruses has revolutionized our understanding of molecular virology and aspects of viral tropism and transmission. Reporter viruses have also enabled the development of high-throughput assays to screen antiviral compounds and antibodies and to perform neutralization assays. However, there remain technical challenges with the design of replication-competent reporter viruses, and each reporter has unique advantages and disadvantages for specific applications. This review describes currently available reporters, design strategies for incorporating reporters into replication-competent paramyxoviruses and orthomyxoviruses, and the variety of applications for which these tools can be utilized both in vitro and in vivo. PMID:27527209

  8. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells.

    PubMed

    Zhao, Chun-Peng; Guo, Xiao; Chen, Si-Jia; Li, Chang-Zheng; Yang, Yun; Zhang, Jun-He; Chen, Shao-Nan; Jia, Yan-Long; Wang, Tian-Yun

    2017-02-20

    Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes.

  9. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells

    PubMed Central

    Zhao, Chun-Peng; Guo, Xiao; Chen, Si-Jia; Li, Chang-Zheng; Yang, Yun; Zhang, Jun-He; Chen, Shao-Nan; Jia, Yan-Long; Wang, Tian-Yun

    2017-01-01

    Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes. PMID:28216629

  10. In vivo cell tracking with bioluminescence imaging.

    PubMed

    Kim, Jung Eun; Kalimuthu, Senthilkumar; Ahn, Byeong-Cheol

    2015-03-01

    Molecular imaging is a fast growing biomedical research that allows the visual representation, characterization and quantification of biological processes at the cellular and subcellular levels within intact living organisms. In vivo tracking of cells is an indispensable technology for development and optimization of cell therapy for replacement or renewal of damaged or diseased tissue using transplanted cells, often autologous cells. With outstanding advantages of bioluminescence imaging, the imaging approach is most commonly applied for in vivo monitoring of transplanted stem cells or immune cells in order to assess viability of administered cells with therapeutic efficacy in preclinical small animal models. In this review, a general overview of bioluminescence is provided and recent updates of in vivo cell tracking using the bioluminescence signal are discussed.

  11. Optimisation of acquisition time in bioluminescence imaging

    NASA Astrophysics Data System (ADS)

    Taylor, Shelley L.; Mason, Suzannah K. G.; Glinton, Sophie; Cobbold, Mark; Styles, Iain B.; Dehghani, Hamid

    2015-03-01

    Decreasing the acquisition time in bioluminescence imaging (BLI) and bioluminescence tomography (BLT) will enable animals to be imaged within the window of stable emission of the bioluminescent source, a higher imaging throughput and minimisation of the time which an animal is anaesthetised. This work investigates, through simulation using a heterogeneous mouse model, two methods of decreasing acquisition time: 1. Imaging at fewer wavelengths (a reduction from five to three); and 2. Increasing the bandwidth of filters used for imaging. The results indicate that both methods are viable ways of decreasing the acquisition time without a loss in quantitative accuracy. Importantly, when choosing imaging wavelengths, the spectral attenuation of tissue and emission spectrum of the source must be considered, in order to choose wavelengths at which a high signal can be achieved. Additionally, when increasing the bandwidth of the filters used for imaging, the bandwidth must be accounted for in the reconstruction algorithm.

  12. Detection of bacteria with bioluminescent reporter bacteriophage.

    PubMed

    Klumpp, Jochen; Loessner, Martin J

    2014-01-01

    Bacteriophages are viruses that exclusively infect bacteria. They are ideally suited for the development of highly specific diagnostic assay systems. Bioluminescent reporter bacteriophages are designed and constructed by integration of a luciferase gene in the virus genome. Relying on the host specificity of the phage, the system enables rapid, sensitive, and specific detection of bacterial pathogens. A bioluminescent reporter phage assay is superior to any other molecular detection method, because gene expression and light emission are dependent on an active metabolism of the bacterial cell, and only viable cells will yield a signal. In this chapter we introduce the concept of creating reporter phages, discuss their advantages and disadvantages, and illustrate the advances made in developing such systems for different Gram-negative and Gram-positive pathogens. The application of bioluminescent reporter phages for the detection of foodborne pathogens is emphasized.

  13. Bioluminescent imaging of bacteria during mouse infection.

    PubMed

    Warawa, Jonathan M; Lawrenz, Matthew B

    2014-01-01

    Diagnostic imaging is a powerful tool that has recently been applied towards the study of infectious diseases. Optical imaging of bioluminescently labeled bacteria in infected animals allows for real-time analysis of bacterial proliferation and dissemination during infection without sacrificing the animal. Imaging also allows for tracking of disease progression in an individual subject over time, has the potential to reveal previously overlooked sites of infection, and reduces the number of research animals used in pathogenesis studies. Here, we describe the use of a deep-cooled CCD camera imager to record light emitted from bacteria during infection. We also describe the process of correlating bioluminescence to bacterial numbers by ex vivo imaging of necropsied tissues. Together these techniques can be used to estimate bacterial burdens in host tissues both in vivo and ex vivo using bioluminescent imaging.

  14. Monitoring of environmental pollutants by bioluminescent bacteria.

    PubMed

    Girotti, Stefano; Ferri, Elida Nora; Fumo, Maria Grazia; Maiolini, Elisabetta

    2008-02-04

    This review deals with the applications of bioluminescent bacteria to the environmental analyses, published during the years 2000-2007. The ecotoxicological assessment, by bioassays, of the environmental risks and the luminescent approaches are reported. The review includes a brief introduction to the characteristics and applications of bioassays, a description of the characteristics and applications of natural bioluminescent bacteria (BLB), and a collection of the main applications to organic and inorganic pollutants. The light-emitting genetically modified bacteria applications, as well as the bioluminescent immobilized systems and biosensors are outlined. Considerations about commercially available BLB and BLB catalogues are also reported. Most of the environmental applications, here mentioned, of luminescent organisms are on wastewater, seawater, surface and ground water, tap water, soil and sediments, air. Comparison to other bioindicators and bioassay has been also made. Various tables have been inserted, to make easier to take a rapid glance at all possible references concerning the topic of specific interest.

  15. Effects of AC/DC magnetic fields, frequency, and nanoparticle aspect ratio on cellular transfection of gene vectors

    NASA Astrophysics Data System (ADS)

    Ford, Kris; Mair, Lamar; Fisher, Mike; Rowshon Alam, Md.; Juliano, Rudolph; Superfine, Richard

    2008-10-01

    In order to make non-viral gene delivery a useful tool in the study and treatment of genetic disorders, it is imperative that these methodologies be further refined to yield optimal results. Transfection of magnetic nanoparticles and nanorods are used as non-viral gene vectors to transfect HeLa EGFP-654 cells that stably express a mutated enhanced green fluorescent protein (EGFP) gene. We deliver antisense oligonucleotides to these cells designed to correct the aberrant splicing caused by the mutation in the EGFP gene. We also transfect human bronchial endothelial cells and immortalized WI-38 lung cells with pEGFP-N1 vectors. To achieve this we bind the genes to magnetic nanoparticles and nanorods and introduce magnetic fields to effect transfection. We wish to examine the effects of magnetic fields on the transfection of these particles and the benefits of using alternating (AC) magnetic fields in improving transfection rates over direct (DC) magnetic fields. We specifically look at the frequency dependence of the AC field and particle aspect ratio as it pertains to influencing transfection rate. We posit that the increase in angular momentum brought about by the AC field and the high aspect ratio of the nanorod particles, is vital to generating the force needed to move the particle through the cell membrane.

  16. A Multichannel Bioluminescence Determination Platform for Bioassays.

    PubMed

    Kim, Sung-Bae; Naganawa, Ryuichi

    2016-01-01

    The present protocol introduces a multichannel bioluminescence determination platform allowing a high sample throughput determination of weak bioluminescence with reduced standard deviations. The platform is designed to carry a multichannel conveyer, an optical filter, and a mirror cap. The platform enables us to near-simultaneously determine ligands in multiple samples without the replacement of the sample tubes. Furthermore, the optical filters beneath the multichannel conveyer are designed to easily discriminate colors during assays. This optical system provides excellent time- and labor-efficiency to users during bioassays.

  17. A novel luciferase fusion protein for highly sensitive optical imaging: from single-cell analysis to in vivo whole-body bioluminescence imaging.

    PubMed

    Mezzanotte, Laura; Blankevoort, Vicky; Löwik, Clemens W G M; Kaijzel, Eric L

    2014-09-01

    Fluorescence and bioluminescence imaging have different advantages and disadvantages depending on the application. Bioluminescence imaging is now the most sensitive optical technique for tracking cells, promoter activity studies, or for longitudinal in vivo preclinical studies. Far-red and near-infrared fluorescence imaging have the advantage of being suitable for both ex vivo and in vivo analysis and have translational potential, thanks to the availability of very sensitive imaging instrumentation. Here, we report the development and validation of a new luciferase fusion reporter generated by the fusion of the firefly luciferase Luc2 to the far-red fluorescent protein TurboFP635 by a 14-amino acid linker peptide. Expression of the fusion protein, named TurboLuc, was analyzed in human embryonic kidney cells, (HEK)-293 cells, via Western blot analysis, fluorescence microscopy, and in vivo optical imaging. The created fusion protein maintained the characteristics of the original bioluminescent and fluorescent protein and showed no toxicity when expressed in living cells. To assess the sensitivity of the reporter for in vivo imaging, transfected cells were subcutaneously injected in animals. Detection limits of cells were 5 × 10(3) and 5 × 10(4) cells for bioluminescent and fluorescent imaging, respectively. In addition, hydrodynamics-based in vivo gene delivery using a minicircle vector expressing TurboLuc allowed for the analysis of luminescent signals over time in deep tissue. Bioluminescence could be monitored for over 30 days in the liver of animals. In conclusion, TurboLuc combines the advantages of both bioluminescence and fluorescence and allows for highly sensitive optical imaging ranging from single-cell analysis to in vivo whole-body bioluminescence imaging.

  18. Effect of mouse VEGF164 on the viability of hydroxyethyl methacrylate-methyl methacrylate-microencapsulated cells in vivo: bioluminescence imaging.

    PubMed

    Cheng, Dangxiao; Lo, Chuen; Sefton, Michael V

    2008-11-01

    Bioluminescent imaging was used to track the viability of luciferase transfected L929 cells in poly(hydroxyethyl methacrylate-co-methyl methacrylate) (HEMA-MMA) microcapsules. Bioluminescence, as determined by Xenogen imaging after addition of luciferin to microcapsules in vitro, increased with time, consistent with an increase in cell number. Capsules were suspended in Matrigel and injected subcutaneously. The bioluminesence in vivo increased over the first 3 weeks and then decreased, both with and without the delivery of mVEGF(164) (1.2 ng/24 h/200 microcapsules in vitro); VEGF delivery was from microencapsulated doubly transfected cells (both luciferase and mVEGF(164)). VEGF delivery was sufficient to generate a greater number of vascular structures, but this did not result in the expected increase in microencapsulated cell viability. Interestingly, the number of vessels at day 28 was less than at day 21, consistent with what would be an expected reduction in VEGF secretion when cell viability is lost. The results presented here do not support the hypothesis that transfection of microencapsulated cells with VEGF is sufficient to correct the oxygen transport limitation, at least with this type of tissue engineering construct. On the other hand, bioluminescent imaging proved to be a useful method of monitoring microencapsulated cell viability over many weeks in vivo.

  19. Efficacy assessment of sustained intraperitoneal paclitaxel therapy in a murine model of ovarian cancer using bioluminescent imaging.

    PubMed

    Vassileva, V; Moriyama, E H; De Souza, R; Grant, J; Allen, C J; Wilson, B C; Piquette-Miller, M

    2008-12-16

    We evaluated the pre-clinical efficacy of a novel intraperitoneal (i.p.) sustained-release paclitaxel formulation (PTX(ePC)) using bioluminescent imaging (BLI) in the treatment of ovarian cancer. Human ovarian carcinoma cells stably expressing the firefly luciferase gene (SKOV3(Luc)) were injected i.p. into SCID mice. Tumour growth was evaluated during sustained or intermittent courses of i.p. treatment with paclitaxel (PTX). In vitro bioluminescence strongly correlated with cell survival and cytotoxicity. Bioluminescent imaging detected tumours before their macroscopic appearance and strongly correlated with tumour weight and survival. As compared with intermittent therapy with Taxol, sustained PTX(ePC) therapy resulted in significant reduction of tumour proliferation, weight and BLI signal intensity, enhanced apoptosis and increased survival times. Our results demonstrate that BLI is a useful tool in the pre-clinical evaluation of therapeutic interventions for ovarian cancer. Moreover, these results provide evidence of enhanced therapeutic efficacy with the sustained PTX(ePC) implant system, which could potentially translate into successful clinical outcomes.

  20. Computation of mixing in large stably stratified enclosures

    NASA Astrophysics Data System (ADS)

    Zhao, Haihua

    This dissertation presents a set of new numerical models for the mixing and heat transfer problems in large stably stratified enclosures. Basing on these models, a new computer code, BMIX++ (Berkeley mechanistic MIXing code in C++), was developed by Christensen (2001) and the author. Traditional lumped control volume methods and zone models cannot model the detailed information about the distributions of temperature, density, and pressure in enclosures and therefore can have significant errors. 2-D and 3-D CFD methods require very fine grid resolution to resolve thin substructures such as jets, wall boundaries, yet such fine grid resolution is difficult or impossible to provide due to computational expense. Peterson's scaling (1994) showed that stratified mixing processes in large stably stratified enclosures can be described using one-dimensional differential equations, with the vertical transport by free and wall jets modeled using standard integral techniques. This allows very large reductions in computational effort compared to three-dimensional numerical modeling of turbulent mixing in large enclosures. The BMIX++ code was developed to implement the above ideas. The code uses a Lagrangian approach to solve 1-D transient governing equations for the ambient fluid and uses analytical models or 1-D integral models to compute substructures. 1-D transient conduction model for the solid boundaries, pressure computation and opening models are also included to make the code more versatile. The BMIX++ code was implemented in C++ and the Object-Oriented-Programming (OOP) technique was intensively used. The BMIX++ code was successfully applied to different types of mixing problems such as stratification in a water tank due to a heater inside, water tank exchange flow experiment simulation, early stage building fire analysis, stratification produced by multiple plumes, and simulations for the UCB large enclosure experiments. Most of these simulations gave satisfying

  1. Stably Expressed Genes Involved in Basic Cellular Functions

    PubMed Central

    Wang, Kejian; Fuscoe, James C.

    2017-01-01

    Stably Expressed Genes (SEGs) whose expression varies within a narrow range may be involved in core cellular processes necessary for basic functions. To identify such genes, we re-analyzed existing RNA-Seq gene expression profiles across 11 organs at 4 developmental stages (from immature to old age) in both sexes of F344 rats (n = 4/group; 320 samples). Expression changes (calculated as the maximum expression / minimum expression for each gene) of >19000 genes across organs, ages, and sexes ranged from 2.35 to >109-fold, with a median of 165-fold. The expression of 278 SEGs was found to vary ≤4-fold and these genes were significantly involved in protein catabolism (proteasome and ubiquitination), RNA transport, protein processing, and the spliceosome. Such stability of expression was further validated in human samples where the expression variability of the homologous human SEGs was significantly lower than that of other genes in the human genome. It was also found that the homologous human SEGs were generally less subject to non-synonymous mutation than other genes, as would be expected of stably expressed genes. We also found that knockout of SEG homologs in mouse models was more likely to cause complete preweaning lethality than non-SEG homologs, corroborating the fundamental roles played by SEGs in biological development. Such stably expressed genes and pathways across life-stages suggest that tight control of these processes is important in basic cellular functions and that perturbation by endogenous (e.g., genetics) or exogenous agents (e.g., drugs, environmental factors) may cause serious adverse effects. PMID:28125669

  2. A spectral model of stably stratified surface-layer turbulence

    NASA Astrophysics Data System (ADS)

    Segalini, A.; Arnqvist, J.; Carlén, I.; Bergström, H.; Alfredsson, P. H.

    2015-06-01

    A new model to determine the spectral velocity tensor in a stably stratified flow is proposed. This model is complementary to the Mann model as it solves the stratified inviscid Rapid Distortion Theory equations analytically, allowing for the determination of the single and two-point velocity spectra as well as the temperature-velocity cross-spectra. The model has been here calibrated and validated against field measurements conducted over a forested area with measurements up to 140 m, therefore covering a region of interest for wind-energy applications.

  3. Bioluminescent bioreporter sensing of foodborne toxins

    NASA Astrophysics Data System (ADS)

    Fraley, Amanda C.; Ripp, Steven; Sayler, Gary S.

    2004-06-01

    Histamine is the primary etiological agent in the foodborne disease scombrotoxicosis, one of the most common food toxicities related to fish consumption. Procedures for detecting histamine in fish products are available, but are often too expensive or too complex for routine use. As an alternative, a bacterial bioluminescent bioreporter has been constructed to develop a biosensor system that autonomously responds to low levels of histamine. The bioreporter contains a promoterless Photorhabdus luminescens lux operon (luxCDABE) fused with the Vibrio anguillarum angR regulatory gene promoter of the anguibactin biosynthetic operon. The bioreporter emitted 1.46 times more bioluminescence than background, 30 minutes after the addition of 100mM histamine. However, specificity was not optimal, as this biosensor generated significant bioluminescence in the presence of L-proline and L-histidine. As a means towards improving histamine specificity, the promoter region of a histamine oxidase gene from Arthrobacter globiformis was cloned upstream of the promotorless lux operon from Photorhabdus luminescens. This recently constructed whole-cell, lux-based bioluminescent bioreporter is currently being tested for optimal performance in the presence of histamine in order to provide a rapid, simple, and inexpensive model sensor for the detection of foodborne toxins.

  4. Bioluminescence: from chemical bonds to photons.

    PubMed

    Hastings, J W

    1975-01-01

    The biological transformation of chemical to photic energy involves an enzyme-mediated chemiluminescent reaction, in which one of the products exists in an electronically excited state, emitting a photon as it returns to the ground state. The colour of bioluminescence differs in different organisms, ranging from the deep blue (460 nm) of certain crustacea, through the bluish green (490 nm) of some bacteria, the green (530 nm) of mushrooms to the red (about 600 nm) of the railroad worm. In one case, energy transfer has been demonstrated from the enzyme system to material that emits light with a longer wavelength. The energies involved range from about 165 to 250 kJ/einstein (40 to 60 kcal/einstein). Boyle first showed that air was involved in bioluminescence in 1668 in his experiments with an air pump. Over the past 100 years, it has become clear that most if not all bioluminescent systems require molecular oxygen. The recent isolation and characterization of an oxygen-containing (peroxide) enzyme intermediate from the bacterial system is described and a reaction mechanism is postulated. This scheme is compared with other hypothetical mechanisms, in particular those involving a four-membered ring intermediate, a dioxetane, in which the simultaneous cleavage of two bonds leaves one product in an excited state. I shall discuss the special role of luciferases in bioluminescence, especially in flashing mechanisms involving 'precharged' intermediates.

  5. High-Resolution Measurements of Coastal Bioluminescence

    DTIC Science & Technology

    2006-09-30

    seen at the canyon edge. The bioluminescence signal confirms that this is biological, and likely a swarm of krill , which it also detects high levels...lifesci.ucsb.edu/~biolum/ Invited talks, Outreach articles: Sep. 2006. Science Year 2007. Photos and research discussion in Worldbook supplement

  6. Multicolor Bioluminescence Obtained Using Firefly Luciferin.

    PubMed

    Kiyama, Masahiro; Saito, Ryohei; Iwano, Satoshi; Obata, Rika; Niwa, Haruki; Maki, Shojiro A

    2016-01-01

    Firefly bioluminescence is widely used in life science research as a useful analysis tool. For example, the adenosine-5`-triphosphate (ATP)-dependent enzymatic firefly bioluminescence reaction has long been utilized as a microbial monitoring tool. Rapid and sensitive firefly luciferin-luciferase combinations are used not only to measure cell viability but also for reporter-gene assays. Recently, bioluminescence was utilized as a noninvasive, real-time imaging tool for living subjects to monitor cells and biological events. However, the number of commercialized luciferase genes is limited and tissue-permeable near-infrared (NIR) region emitting light is required for in vivo imaging. In this review, recent studies describing synthetic luciferin analogues predicted to have red-shifted bioluminescence are summarized. Luciferase substrates emitting red, green, and blue light that were designed and developed in our laboratory are presented. The longest emission wavelength of the synthesized luciferin analogues was recorded at 675 nm, which is within the NIR region. This compound is now commercially available as "Aka Lumine®".

  7. Bioluminescence for determining energy state of plants

    NASA Technical Reports Server (NTRS)

    Ching, T. M.

    1975-01-01

    Bioluminescence produced by the luciferin-luciferase system is a very sensitive assay for ATP content in extracts of plant materials. The ATP test for seed and pollen viability and vigor is presented, along with prediction of high growth potential and productivity in new crosses and selections of breeding materials. ATP as an indicator for environmental quality, stresses, and metabolic regulation is also considered.

  8. Bioluminescence lights the way to food safety

    NASA Astrophysics Data System (ADS)

    Brovko, Lubov Y.; Griffiths, Mansel W.

    2003-07-01

    The food industry is increasingly adopting food safety and quality management systems that are more proactive and preventive than those used in the past which have tended to rely on end product testing and visual inspection. The regulatory agencies in many countries are promoting one such management tool, Hazard Analysis Critical Control Point (HACCP), as a way to achieve a safer food supply and as a basis for harmonization of trading standards. Verification that the process is safe must involve microbiological testing but the results need not be generated in real-time. Of all the rapid microbiological tests currently available, the only ones that come close to offering real-time results are bioluminescence-based methods. Recent developments in application of bioluminescence for food safety issues are presented in the paper. These include the use of genetically engineered microorganisms with bioluminescent and fluorescent phenotypes as a real time indicator of physiological state and survival of food-borne pathogens in food and food processing environments as well as novel bioluminescent-based methods for rapid detection of pathogens in food and environmental samples. Advantages and pitfalls of the methods are discussed.

  9. Bioluminescence imaging of bone metastasis in rodents.

    PubMed

    Snoeks, Thomas J A; van Beek, Ermond; Que, Ivo; Kaijzel, Eric L; Löwik, Clemens W G M

    2012-01-01

    Optical imaging is a valuable technique for visualizing and quantifying biological processes in living -organisms. Optical imaging can be divided into two main imaging modalities: bioluminescence imaging and fluorescence imaging. This chapter describes the use of these imaging techniques to image tumour cells in mouse models of cancer and to detect early bone metastasis.

  10. Bioluminescent bioreporter integrated circuit detection methods

    SciTech Connect

    Simpson, Michael L.; Paulus, Michael J.; Sayler, Gary S.; Applegate, Bruce M.; Ripp, Steven A.

    2005-06-14

    Disclosed are monolithic bioelectronic devices comprising a bioreporter and an OASIC. These bioluminescent bioreporter integrated circuit are useful in detecting substances such as pollutants, explosives, and heavy-metals residing in inhospitable areas such as groundwater, industrial process vessels, and battlefields. Also disclosed are methods and apparatus for detection of particular analytes, including ammonia and estrogen compounds.

  11. Amplitude Metrics for Cellular Circadian Bioluminescence Reporters

    PubMed Central

    St. John, Peter C.; Taylor, Stephanie R.; Abel, John H.; Doyle, Francis J.

    2014-01-01

    Bioluminescence rhythms from cellular reporters have become the most common method used to quantify oscillations in circadian gene expression. These experimental systems can reveal phase and amplitude change resulting from circadian disturbances, and can be used in conjunction with mathematical models to lend further insight into the mechanistic basis of clock amplitude regulation. However, bioluminescence experiments track the mean output from thousands of noisy, uncoupled oscillators, obscuring the direct effect of a given stimulus on the genetic regulatory network. In many cases, it is unclear whether changes in amplitude are due to individual changes in gene expression level or to a change in coherence of the population. Although such systems can be modeled using explicit stochastic simulations, these models are computationally cumbersome and limit analytical insight into the mechanisms of amplitude change. We therefore develop theoretical and computational tools to approximate the mean expression level in large populations of noninteracting oscillators, and further define computationally efficient amplitude response calculations to describe phase-dependent amplitude change. At the single-cell level, a mechanistic nonlinear ordinary differential equation model is used to calculate the transient response of each cell to a perturbation, whereas population-level dynamics are captured by coupling this detailed model to a phase density function. Our analysis reveals that amplitude changes mediated at either the individual-cell or the population level can be distinguished in tissue-level bioluminescence data without the need for single-cell measurements. We demonstrate the effectiveness of the method by modeling experimental bioluminescence profiles of light-sensitive fibroblasts, reconciling the conclusions of two seemingly contradictory studies. This modeling framework allows a direct comparison between in vitro bioluminescence experiments and in silico ordinary

  12. Amplitude metrics for cellular circadian bioluminescence reporters.

    PubMed

    St John, Peter C; Taylor, Stephanie R; Abel, John H; Doyle, Francis J

    2014-12-02

    Bioluminescence rhythms from cellular reporters have become the most common method used to quantify oscillations in circadian gene expression. These experimental systems can reveal phase and amplitude change resulting from circadian disturbances, and can be used in conjunction with mathematical models to lend further insight into the mechanistic basis of clock amplitude regulation. However, bioluminescence experiments track the mean output from thousands of noisy, uncoupled oscillators, obscuring the direct effect of a given stimulus on the genetic regulatory network. In many cases, it is unclear whether changes in amplitude are due to individual changes in gene expression level or to a change in coherence of the population. Although such systems can be modeled using explicit stochastic simulations, these models are computationally cumbersome and limit analytical insight into the mechanisms of amplitude change. We therefore develop theoretical and computational tools to approximate the mean expression level in large populations of noninteracting oscillators, and further define computationally efficient amplitude response calculations to describe phase-dependent amplitude change. At the single-cell level, a mechanistic nonlinear ordinary differential equation model is used to calculate the transient response of each cell to a perturbation, whereas population-level dynamics are captured by coupling this detailed model to a phase density function. Our analysis reveals that amplitude changes mediated at either the individual-cell or the population level can be distinguished in tissue-level bioluminescence data without the need for single-cell measurements. We demonstrate the effectiveness of the method by modeling experimental bioluminescence profiles of light-sensitive fibroblasts, reconciling the conclusions of two seemingly contradictory studies. This modeling framework allows a direct comparison between in vitro bioluminescence experiments and in silico ordinary

  13. Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA

    SciTech Connect

    Matsushima, H.; Bogenmann, E. )

    1990-09-01

    Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment.

  14. Bioluminescence as an ecological factor during high Arctic polar night

    PubMed Central

    Cronin, Heather A.; Cohen, Jonathan H.; Berge, Jørgen; Johnsen, Geir; Moline, Mark A.

    2016-01-01

    Bioluminescence commonly influences pelagic trophic interactions at mesopelagic depths. Here we characterize a vertical gradient in structure of a generally low species diversity bioluminescent community at shallower epipelagic depths during the polar night period in a high Arctic fjord with in situ bathyphotometric sampling. Bioluminescence potential of the community increased with depth to a peak at 80 m. Community composition changed over this range, with an ecotone at 20–40 m where a dinoflagellate-dominated community transitioned to dominance by the copepod Metridia longa. Coincident at this depth was bioluminescence exceeding atmospheric light in the ambient pelagic photon budget, which we term the bioluminescence compensation depth. Collectively, we show a winter bioluminescent community in the high Arctic with vertical structure linked to attenuation of atmospheric light, which has the potential to influence pelagic ecology during the light-limited polar night. PMID:27805028

  15. Bioluminescence as an ecological factor during high Arctic polar night.

    PubMed

    Cronin, Heather A; Cohen, Jonathan H; Berge, Jørgen; Johnsen, Geir; Moline, Mark A

    2016-11-02

    Bioluminescence commonly influences pelagic trophic interactions at mesopelagic depths. Here we characterize a vertical gradient in structure of a generally low species diversity bioluminescent community at shallower epipelagic depths during the polar night period in a high Arctic fjord with in situ bathyphotometric sampling. Bioluminescence potential of the community increased with depth to a peak at 80 m. Community composition changed over this range, with an ecotone at 20-40 m where a dinoflagellate-dominated community transitioned to dominance by the copepod Metridia longa. Coincident at this depth was bioluminescence exceeding atmospheric light in the ambient pelagic photon budget, which we term the bioluminescence compensation depth. Collectively, we show a winter bioluminescent community in the high Arctic with vertical structure linked to attenuation of atmospheric light, which has the potential to influence pelagic ecology during the light-limited polar night.

  16. Bioluminescence microscopy using a short focal-length imaging lens.

    PubMed

    Ogoh, K; Akiyoshi, R; May-Maw-Thet; Sugiyama, T; Dosaka, S; Hatta-Ohashi, Y; Suzuki, H

    2014-03-01

    Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low-light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera. As bioluminescence microscopy requires no excitation light, it lacks the photo-toxicity associated with fluorescence imaging and permits the long-term, nonlethal observation of living cells. Thus, bioluminescence microscopy would be a powerful tool in cellular biology that complements fluorescence microscopy.

  17. Bioluminescence as an ecological factor during high Arctic polar night

    NASA Astrophysics Data System (ADS)

    Cronin, Heather A.; Cohen, Jonathan H.; Berge, Jørgen; Johnsen, Geir; Moline, Mark A.

    2016-11-01

    Bioluminescence commonly influences pelagic trophic interactions at mesopelagic depths. Here we characterize a vertical gradient in structure of a generally low species diversity bioluminescent community at shallower epipelagic depths during the polar night period in a high Arctic fjord with in situ bathyphotometric sampling. Bioluminescence potential of the community increased with depth to a peak at 80 m. Community composition changed over this range, with an ecotone at 20–40 m where a dinoflagellate-dominated community transitioned to dominance by the copepod Metridia longa. Coincident at this depth was bioluminescence exceeding atmospheric light in the ambient pelagic photon budget, which we term the bioluminescence compensation depth. Collectively, we show a winter bioluminescent community in the high Arctic with vertical structure linked to attenuation of atmospheric light, which has the potential to influence pelagic ecology during the light-limited polar night.

  18. Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature.

    PubMed Central

    Zhou, Z; Gong, Q; Ye, B; Fan, Z; Makielski, J C; Robertson, G A; January, C T

    1998-01-01

    We have established stably transfected HEK 293 cell lines expressing high levels of functional human ether-a go-go-related gene (HERG) channels. We used these cells to study biochemical characteristics of HERG protein, and to study electrophysiological and pharmacological properties of HERG channel current at 35 degrees C. HERG-transfected cells expressed an mRNA band at 4.0 kb. Western blot analysis showed two protein bands (155 and 135 kDa) slightly larger than the predicted molecular mass (127 kDa). Treatment with N-glycosidase F converted both bands to smaller molecular mass, suggesting that both are glycosylated, but at different levels. HERG current activated at voltages positive to -50 mV, maximum current was reached with depolarizing steps to -10 mV, and the current amplitude declined at more positive voltages, similar to HERG channel current expressed in other heterologous systems. Current density at 35 degrees C, compared with 23 degrees C, was increased by more than twofold to a maximum of 53.4 +/- 6.5 pA/pF. Activation, inactivation, recovery from inactivation, and deactivation kinetics were rapid at 35 degrees C, and more closely resemble values reported for the rapidly activating delayed rectifier K+ current (I(Kr)) at physiological temperatures. HERG channels were highly selective for K+. When we used an action potential clamp technique, HERG current activation began shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude during phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potential, and deactivation of HERG current could participate in phase 4 depolarization. HERG current was blocked by low concentrations of E-4031 (IC50 7.7 nM), a value close to that reported for I(Kr) in native cardiac myocytes. Our data support the postulate that HERG encodes a major constituent of I(Kr) and suggest that

  19. Transformation Experiment Using Bioluminescence Genes of "Vibrio fischeri."

    ERIC Educational Resources Information Center

    Slock, James

    1995-01-01

    Bioluminescence transformation experiments show students the excitement and power of recombinant DNA technology. This laboratory experiment utilizes two plasmids of "Vibrio fischeri" in a transformation experiment. (LZ)

  20. Bioluminescent assay for human lymphocyte blast transformation.

    PubMed

    Bulanova, E G; Budagyan, V M; Romanova, N A; Brovko LYu; Ugarova, N N

    1995-05-01

    One of the basic tests of in vitro evaluation of immune cell functional activity is a proliferative response of lymphocytes on the action of external stimuli such as mitogenic lectines, antigens, etc. We compared two methods used to assess the lymphocyte functional status. (1) [3H]thymidine incorporation and (2) bioluminescence for determination of intracellular ATP in blast cells. Comparison has been done for healthy donors and patients with proven low immunological status. The proposed bioluminescent method for evaluation of the proliferative response was shown to be sensitive enough for diagnostic purposes. This method allows one to process a large number of samples at the same time and correlates highly with the radionuclide test use hazardous radioactive materials.

  1. Bioluminescence imaging of myeloperoxidase activity in vivo

    PubMed Central

    Gross, Shimon; Gammon, Seth T; Moss, Britney L; Rauch, Daniel; Harding, John; Heinecke, Jay W; Ratner, Lee; Piwnica-Worms, David

    2010-01-01

    The myeloperoxidase (MPO) system of activated phagocytes is central to normal host defense mechanisms, and dysregulated MPO contributes to the pathogenesis of inflammatory disease states ranging from atherosclerosis to cancer. Here we show that upon systemic administration, the small molecule luminol enables noninvasive bioluminescence imaging (BLI) of MPO activity in vivo. Luminol-BLI allowed quantitative longitudinal monitoring of MPO activity in animal models of acute dermatitis, mixed allergic contact hypersensitivity, focal arthritis and spontaneous large granular lymphocytic tumors. Bioluminescence colocalized with histological sites of inflammation and was totally abolished in gene-deleted Mpo−/− mice, despite massive tissue infiltration of neutrophils and activated eosinophils, indicating that eosinophil peroxidase did not contribute to luminol-BLI in vivo. Thus, luminol-BLI provides a noninvasive, specific and highly sensitive optical readout of phagocyte-mediated MPO activity in vivo and may enable new diagnostic applications in a wide range of acute and chronic inflammatory conditions. PMID:19305414

  2. Analysis of river water by bioluminescent biotests.

    PubMed

    Kuznetsov, A M; Rodicheva, E K; Medvedeva, S E

    1999-01-01

    The bacterial bioluminescence has high sensitivity to the action of various inhibitors of biological activity. The lyophilized luminous bacteria Photobacterium phosphoreum (Microbiosensor B17 677F) and luminous strain Escherichia coli (Microbiosensor EC) from the Culture Collection IBSO were used to create bioluminescent biotests. They have been applied in ecological monitoring to determine the overall toxicity of the Yenisei and Angara Rivers and some water sources of Altai Territory. As a rule the heaviest pollution of water in studied rivers was registered near cities and settlements. The luminous bacteria biotests are simple and convenient in work, standardized and quantitative, have rapid response to actions of different substances and high sensitivity to environmental pollutants. It takes less than 30 min to do the biotest (the other biotests take 48--96 h).

  3. Bioluminescence imaging of myeloperoxidase activity in vivo.

    PubMed

    Gross, Shimon; Gammon, Seth T; Moss, Britney L; Rauch, Daniel; Harding, John; Heinecke, Jay W; Ratner, Lee; Piwnica-Worms, David

    2009-04-01

    The myeloperoxidase (MPO) system of activated phagocytes is central to normal host defense mechanisms, and dysregulated MPO contributes to the pathogenesis of inflammatory disease states ranging from atherosclerosis to cancer. Here we show that upon systemic administration, the small molecule luminol enables noninvasive bioluminescence imaging (BLI) of MPO activity in vivo. Luminol-BLI allowed quantitative longitudinal monitoring of MPO activity in animal models of acute dermatitis, mixed allergic contact hypersensitivity, focal arthritis and spontaneous large granular lymphocytic tumors. Bioluminescence colocalized with histological sites of inflammation and was totally abolished in gene-deleted Mpo(-/-) mice, despite massive tissue infiltration of neutrophils and activated eosinophils, indicating that eosinophil peroxidase did not contribute to luminol-BLI in vivo. Thus, luminol-BLI provides a noninvasive, specific and highly sensitive optical readout of phagocyte-mediated MPO activity in vivo and may enable new diagnostic applications in a wide range of acute and chronic inflammatory conditions.

  4. Polymer Drag Reduction and Bioluminescence Reduction

    DTIC Science & Technology

    2007-09-30

    jet to cavitate is also reduced by the presence of trace amounts of polymer. Imaging of bioluminescence stimulated by a turbulent jet will assess...regulated by a computer-controlled pump system located downstream of the pipe. Upstream of the pipe is a tapered nozzle to assure laminar flow at the...inlet even at high flow rates. Flow rate is measured by a mass flow meter downstream of the pump and the pressure drop within the pipe is measured

  5. Bioluminescence for Detection of Trace Compounds

    DTIC Science & Technology

    1990-03-08

    Photobacterium leiognathi and their expression in Escherichia coli, Qene. 54:203-210. Eberhard, A., Burlingame, A.L., Eberhard, C., Kenyon, G.L., Nealson, K.H...1985). The stimulation of bioluminescence in Photobacterium leiognathi as a potential prescreen for antitumor agents. L Antibiotics. 39:1401-1407... Photobacterium leiognatii PL721 ligated into the plasmid pACYCI84 (Figure 2), was selected for this research because of its known response to carcinogens

  6. Ballistic transfection of mammalian cells in vivo

    SciTech Connect

    Kolesnikov, V.A.; Zelenin, A.V.; Zelenina, I.A.

    1995-11-01

    The method of ballistic transfection initially proposed for genetic transformation of plants was used for animal cells in vitro and in situ. The method consists in bombarding the transfected cells with microparticles of heavy metals carrying foreign DNA. Penetrating the cell nucleus, the microparticles transport the introduced gene. Successful genetic transformation of the cultured mouse cells and fish embryos was realized, and this allowed the study of mammalian cells in situ. The performed studies allowed us to demonstrate expression of the reporter genes of chloramphenicol acetyltransferase, galactosidase, and neomycin phosphotransferase in the mouse liver, mammary gland and kidney explants, in the liver and cross-striated muscle of mouse and rat in situ, and in developing mouse embryos at the stages of two-cell embryo, morula, and blastocyst. All these genes were introduced by ballistic transfection. In the liver and cross-striated muscle the transgene activity was detected within two to three months after transfection. Thus, the ballistic introduction of the foreign genes in the cells in situ was demonstrated, and this opens possibilities for the use of this method in gene therapy. Methodical aspects of the bombarding and transfection are considered in detail, and the published data on transfection and genetic transformation of mammalian cells are discussed. 41 refs., 13 figs., 1 tab.

  7. GFP-like proteins stably accumulate in lysosomes.

    PubMed

    Katayama, Hiroyuki; Yamamoto, Akitsugu; Mizushima, Noboru; Yoshimori, Tamotsu; Miyawaki, Atsushi

    2008-01-01

    Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its GFP variants (Aequorea GFPs), and more recently the novel GFP-like proteins from Anthozoa have greatly advanced our technologies for fluorescently labeling cells, organelles, and proteins. It has been shown, however, that some GFP-like proteins have a tendency to oligomerize and aggregate. Transfection of GFP-like proteins into cultured mammalian cells results in bright punctate structures, which are thought to be cytosolic protein aggregates. In this study, we demonstrate that these structures are not cytosolic aggregates but lysosomes that have accumulated the GFP-like proteins. Our biochemical and immunocytochemical experiments have revealed that certain GFP-like proteins expressed in the cytosol enter lysosomes possibly by an autophagy-related mechanism, but retain their fluorescence because of resistance not only to acidity but also to lysosomal proteases.

  8. Mixing efficiency of turbulent patches in stably stratified flows

    NASA Astrophysics Data System (ADS)

    Garanaik, Amrapalli; Venayagamoorthy, Subhas Karan

    2016-11-01

    A key quantity that is essential for estimating the turbulent diapycnal (irreversible) mixing in stably stratified flow is the mixing efficiency Rf*, which is a measure of the amount of turbulent kinetic energy that is irreversibly converted into background potential energy. In particular, there is an ongoing debate in the oceanographic mixing community regarding the utility of the buoyancy Reynolds number (Reb) , particularly with regard to how mixing efficiency and diapycnal diffusivity vary with Reb . Specifically, is there a universal relationship between the intensity of turbulence and the strength of the stratification that supports an unambiguous description of mixing efficiency based on Reb ? The focus of the present study is to investigate the variability of Rf* by considering oceanic turbulence data obtained from microstructure profiles in conjunction with data from laboratory experiments and DNS. Field data analysis has done by identifying turbulent patches using Thorpe sorting method for potential density. The analysis clearly shows that high mixing efficiencies can persist at high buoyancy Reynolds numbers. This is contradiction to previous studies which predict that mixing efficiency should decrease universally for Reb greater than O (100) . Funded by NSF and ONR.

  9. Two phenomenological constants explain similarity laws in stably stratified turbulence.

    PubMed

    Katul, Gabriel G; Porporato, Amilcare; Shah, Stimit; Bou-Zeid, Elie

    2014-02-01

    In stably stratified turbulent flows, the mixing efficiency associated with eddy diffusivity for heat, or equivalently the turbulent Prandtl number (Pr(t)), is fraught with complex dynamics originating from the scalewise interplay between shear generation of turbulence and its dissipation by density gradients. A large corpus of data and numerical simulations agree on a near-universal relation between Pr(t) and the Richardson number (R(i)), which encodes the relative importance of buoyancy dissipation to mechanical production of turbulent kinetic energy. The Pr(t)-R(i) relation is shown to be derivable solely from the cospectral budgets for momentum and heat fluxes if a Rotta-like return to isotropy closure for the pressure-strain effects and Kolmogorov's theory for turbulent cascade are invoked. The ratio of the Kolmogorov to the Kolmogorov-Obukhov-Corrsin phenomenological constants, and a constant associated with isotropization of the production whose value (= 3/5) has been predicted from Rapid Distortion Theory, explain all the macroscopic nonlinearities.

  10. Turbulence comes in bursts in stably stratified flows

    NASA Astrophysics Data System (ADS)

    Rorai, C.; Mininni, P. D.; Pouquet, A.

    2014-04-01

    There is a clear distinction between simple laminar and complex turbulent fluids; however, in some cases, as for the nocturnal planetary boundary layer, a stable and well-ordered flow can develop intense and sporadic bursts of turbulent activity that disappear slowly in time. This phenomenon is ill understood and poorly modeled and yet it is central to our understanding of weather and climate dynamics. We present here data from direct numerical simulations of stratified turbulence on grids of 20483 points that display the somewhat paradoxical result of measurably stronger events for more stable flows, not only in the temperature and vertical velocity derivatives as commonplace in turbulence, but also in the amplitude of the fields themselves, contrary to what happens for homogenous isotropic turbulent flows. A flow visualization suggests that the extreme values take place in Kelvin-Helmoltz overturning events and fronts that develop in the field variables. These results are confirmed by the analysis of a simple model that we present. The model takes into consideration only the vertical velocity and temperature fluctuations and their vertical derivatives. It indicates that in stably stratified turbulence, the stronger bursts can occur when the flow is expected to be more stable. The bursts are generated by a rapid nonlinear amplification of energy stored in waves and are associated with energetic interchanges between vertical velocity and temperature (or density) fluctuations in a range of parameters corresponding to the well-known saturation regime of stratified turbulence.

  11. Stably Doped Conducting Polymer Nanoshells by Surface Initiated Polymerization.

    PubMed

    Li, Junwei; Yoon, Soon Joon; Hsieh, Bao-Yu; Tai, Wanyi; O'Donnell, Matthew; Gao, Xiaohu

    2015-12-09

    Despite broad applications ranging from electronics to biomedical sensing and imaging, a long-standing problem of conducting polymers is the poor resistance to dedoping, which directly affects their signature electrical and optical properties. This problem is particularly significant for biomedical uses because of fast leaching of dopant ions in physiological environments. Here, we describe a new approach to engineer multimodal core-shell nanoparticles with a stably doped conductive polymer shell in biological environments. It was achieved by making a densely packed polymer brush rather than changing its molecular structure. Polyaniline (PANI) was used as a model compound due to its concentrated near-infrared (NIR) absorption. It was grafted onto a magnetic nanoparticle via a polydopamine intermediate layer. Remarkably, at pH 7 its conductivity is ca. 2000× higher than conventional PANI nanoshells. Similarly, its NIR absorption is enhanced by 2 orders of magnitude, ideal for photothermal imaging and therapy. Another surprising finding is its nonfouling property, even outperforming polyethylene glycol. This platform technology is also expected to open exciting opportunities in engineering stable conductive materials for electronics, imaging, and sensing.

  12. A REVIEW OF ENVIRONMENTAL APPLICATIONS OF BIOLUMINESCENCE MEASUREMENTS

    EPA Science Inventory

    This review of the recent literature on environmental applications of bioluminescence systems will focus on in vivo and in vitro bioluminescence methods that have been utilized to elucidate properties of chemicals, toxic and mutagenic effects, and to estimate biomass. The unifyin...

  13. Characterization of bioluminescent derivatives of assimilable organic carbon test bacteria.

    PubMed

    Haddix, Pryce L; Shaw, Nancy J; LeChevallier, Mark W

    2004-02-01

    The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.

  14. Evaluation of the ecotoxicity of pollutants with bioluminescent microorganisms.

    PubMed

    Fernández-Piñas, Francisca; Rodea-Palomares, Ismael; Leganés, Francisco; González-Pleiter, Miguel; Angeles Muñoz-Martín, M

    2014-01-01

    This chapter deals with the use of bioluminescent microorganisms in environmental monitoring, particularly in the assessment of the ecotoxicity of pollutants. Toxicity bioassays based on bioluminescent microorganisms are an interesting complement to classical toxicity assays, providing easiness of use, rapid response, mass production, and cost effectiveness. A description of the characteristics and main environmental applications in ecotoxicity testing of naturally bioluminescent microorganisms, covering bacteria and eukaryotes such as fungi and dinoglagellates, is reported in this chapter. The main features and applications of a wide variety of recombinant bioluminescent microorganisms, both prokaryotic and eukaryotic, are also summarized and critically considered. Quantitative structure-activity relationship models and hormesis are two important concepts in ecotoxicology; bioluminescent microorganisms have played a pivotal role in their development. As pollutants usually occur in complex mixtures in the environment, the use of both natural and recombinant bioluminescent microorganisms to assess mixture toxicity has been discussed. The main information has been summarized in tables, allowing quick consultation of the variety of luminescent organisms, bioluminescence gene systems, commercially available bioluminescent tests, environmental applications, and relevant references.

  15. Single-cell bioluminescence and GFP in biofilm research

    SciTech Connect

    Palmer, R.J. Jr, Sayler, G., White, D.C.; Phiefer, C.

    1996-12-31

    Using flow cells and a combination of microscopy techniques, we can unequivocally identify single bacterial cells that express bioluminescent and fluorescent bioreporters. We have shown that, for attached cells, bioluminescence output within a bacterial strain can vary greatly from cell to cell.

  16. Detection of ATP and NADH: A Bioluminescent Experience.

    ERIC Educational Resources Information Center

    Selig, Ted C.; And Others

    1984-01-01

    Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

  17. Functional identification of the stable transfection C5aR cell line Molt-4.

    PubMed

    Zhang, Chunmei; Xu, Ruonan; Wang, Jianan; Han, Gencheng; Chen, Guojiang; Wang, Renxi; Wei, Huawei; Shen, Beifen; Ma, Yuanfang; Li, Yan

    2007-12-01

    The complement C5 anaphylatoxin receptor is a member of the seven transmembrane-spanning G protein-coupled receptor superfamily that signals through Galphai and Galpha16. C5aR is mostly expressed on neutrophils, macrophages and endothelial cells. C5a and C5aR interaction plays an important role in numerous biological effects such as in vivo cytokine storm which results in inflammatory damage. Considering the limitation of collection of human peripheral blood neutrophils and their short half life, the stably transfected cell line for studying the biological effects of C5aR is needed. In this study, we transfected C5aR gene into Molt-4 cell line and examined the function of ectopic C5aR. Our results showed stable expression of the C5aR in Molt-4 cell line and their interaction with human C5a induced ERK1/2 phosphorylation, Ca++ influx. This stable transfected cell line may provide a useful tool for studying signal pathways related to C5a and C5aR interplay and antibody development specific for C5aR.

  18. Imaging of bubonic plague dynamics by in vivo tracking of bioluminescent Yersinia pestis.

    PubMed

    Nham, Toan; Filali, Sofia; Danne, Camille; Derbise, Anne; Carniel, Elisabeth

    2012-01-01

    Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI) to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba), followed by a colonization of the draining inguinal lymph node(s), and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response.

  19. Optimizacion of Babesia bovis transfection methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The tick borne Babesia parasites remain an important limitation for development of cattle industries worldwide. A stable transfection of Babesia bovis will be useful for functional analysis of the recently sequenced B. bovis genome and to design improved methods to control Babesia infections. In thi...

  20. In Vivo Bioluminescence Imaging of Intratumoral Bacteria.

    PubMed

    Cronin, Michelle; Akin, Ali R; Francis, Kevin P; Tangney, Mark

    2016-01-01

    This chapter describes the use of whole-body bioluminescent imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in therapy of cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and tumor sites concurrently. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor-bearing mice. This powerful, and inexpensive, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research. This protocol outlines the procedure for studying lux-tagged Escherichia coli and Bifidobacterium breve in mice, demonstrating the spatial and temporal readout from 2D and 3D BLI achievable with whole-body in vivo luminescence imaging.

  1. In vitro transfection mediated by dendrigraft poly(L-lysines): the effect of structure and molecule size.

    PubMed

    Hofman, Jakub; Buncek, Martin; Haluza, Radovan; Streinz, Ludvik; Ledvina, Miroslav; Cigler, Petr

    2013-02-01

    Dendritic poly(L-lysines) (DGL) constitute promising nanomaterials applicable as a nonviral gene-delivery vector. In this study, we evaluate the transfection abilities of four DGL generations with special emphasis on the systematic description of the relationship of how generation (i.e., molecule size) affects the transfection efficacy. Using Hep2 cells, we demonstrated that the capability of unmodified DGL to deliver plasmid is of a magnitude lower than that of jetPEI. On the other hand, employing the Hep2 cell line stably transduced with eGFP, we observed that DGL G5 delivers the siRNA oligonucleotide with the same efficiency as Lipofectamine 2000. In further experiments, it was shown that DGL affords excellent ability to bind DNA, protect it against DNase I attack, and internalize it into cells.

  2. Construction of a bioluminescent reporter strain to detect polychlorinated biphenyls

    SciTech Connect

    Layton, A.C.; Muccini, M.; Ghosh, M.M.; Sayler, G.S.

    1998-12-01

    A bioluminescent reporter strain, Ralstonia eutropha ENV307 (pUTK60), was constructed for the detection of polychlorinated biphenyls by inserting the biphenyl promoter upstream of the bioluminescence genes. In the presence of a nonionic surfactant, which enhances the solubility of chlorinated biphenyls, bioluminescence was induced three- to fourfold over background by biphenyl, monochlorinated biphenyls, and Aroclor 1242. The minimum detection limits for these compounds ranged from 0.15 mg/liter for 4-chlorobiphenyl to 1.5 mg/liter for Aroclor 1242.

  3. Effect of targeted ovarian cancer therapy using amniotic fluid mesenchymal stem cells transfected with enhanced green fluorescent protein-human interleukin-2 in vivo.

    PubMed

    You, Qi; Yao, Yuan; Zhang, Yuanlong; Fu, Songbin; Du, Mei; Zhang, Guangmei

    2015-10-01

    The aim of the present study was to investigate the effect of using amniotic fluid mesenchymal stem cells (AF-MSCs) in targeted ovarian cancer therapy in vivo. AF-MSCs were isolated from human second trimester AF and a plasmid, enhanced green fluorescent protein‑human interleukin‑2 (pEGFP‑hIL‑2) was formed. The plasmid was stably transfected into the AF‑MSCs and the cells were intravenously injected into ovarian cancer nude mice models. Following stable transfection of the vector, tumor formation, and the expression and activity of hIL‑2 were investigated, and microscopic pathological examinations of the tumor were performed. It was found that AF‑MSCs exhibited high motility during migration in vivo, and the vector, pEGFP‑hIL‑2 can be stably transfected into AF‑MSCs. Following stable transfection, this type of stem cell is able to successfully transport the therapeutic gene, IL-2, migrate to the ovarian cancer tumor site to secrete the functional IL-2 and treat the tumor. Thus, AF-MSCs may serve as transporters for therapeutic genes targeting ovarian tumor sites and, therefore, be involved in the treatment of tumors.

  4. Measuring IL-1β Processing by Bioluminescence Sensors I: Using a Bioluminescence Resonance Energy Transfer Biosensor.

    PubMed

    Compan, Vincent; Pelegrín, Pablo

    2016-01-01

    IL-1β processing is one of the hallmarks of inflammasome activation and drives the initiation of the inflammatory response. For decades, Western blot or ELISA have been extensively used to study this inflammatory event. Here, we describe the use of a bioluminescence resonance energy transfer (BRET) biosensor to monitor IL-1β processing in real time and in living macrophages either using a plate reader or a microscope.

  5. Transfection of mouse ribosomal DNA into rat cells: faithful transcription and processing.

    PubMed Central

    Vance, V B; Thompson, E A; Bowman, L H

    1985-01-01

    Truncated mouse ribosomal DNA (rDNA) genes were stably incorporated into rat HTC-5 cells by DNA-mediated cell transfection techniques. The mouse rDNA genes were accurately transcribed in these rat cells indicating that there is no absolute species specificity of rDNA transcription between mouse and rat. No more than 170 nucleotides of the 5' nontranscribed spacer was required for the accurate initiation of mouse rDNA transcription in rat cells. Further, the mouse transcripts were accurately cleaved at the 5' end of the 18S rRNA sequence, even though these transcripts contained neither the 3' end of mouse 18S rRNA nor any other downstream mouse sequences. Thus, cleavage at the 5' end of 18S rRNA is not dependent on long range interactions involving these downstream sequences. Images PMID:2997749

  6. Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency

    PubMed Central

    Sork, Helena; Nordin, Joel Z; Turunen, Janne J; Wiklander, Oscar PB; Bestas, Burcu; Zaghloul, Eman M; Margus, Helerin; Padari, Kärt; Duru, Adil D; Corso, Giulia; Bost, Jeremy; Vader, Pieter; Pooga, Margus; Smith, CI Edvard; Wood, Matthew JA; Schiffelers, Raymond M; Hällbrink, Mattias; Andaloussi, Samir EL

    2016-01-01

    The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents. PMID:27111416

  7. Development of Leishmania donovani stably expressing DsRed for flow cytometry-based drug screening using chalcone thiazolyl-hydrazone as a new antileishmanial target.

    PubMed

    Jaiswal, Anil Kumar; Rao, K Bhaskara; Kushwaha, Pragati; Rawat, Keerti; Modukuri, Ram K; Khare, Prashant; Joshi, Sumit; Mishra, Shikha; Rai, Ambak; Sashidhara, Koneni V; Dube, Anuradha

    2016-12-01

    Green fluorescent protein produces significant fluorescence and is extremely stable, however its excitation maximum is close to the ultraviolet range and thus can damage living cells. Hence, Leishmania donovani stably expressing DsRed were developed and their suitability for flow cytometry-based antileishmanial screening was assessed by evaluating the efficacies of standard drugs as well as newly synthesised chalcone thiazolyl-hydrazone compounds. The DsRed gene was successfully integrated at the 18S rRNA locus of L. donovani and transfectants (LdDsRed) were selected using hygromycin B. Enhanced expression of DsRed and a high level of infectivity to J774A.1 macrophages were achieved, which was confirmed by fluorescence microscopy and flow cytometry. Furthermore, these LdDsRed transfectants were utilised for development of an in vitro screening assay using the standard antileishmanial drugs miltefosine, amphotericin B, pentamidine and paromomycin. The response of transfectants to standard drugs correlated well with previous reports. Subsequently, the suitability of this system was further assessed by screening a series of 18 newly synthesised chalcone thiazolyl-hydrazone compounds in vitro for their antileishmanial activity, wherein 8 compounds showed moderate antileishmanial activity. The most active compound 5g, with ca. 73% splenic parasite reduction, exerted its activity via generating nitric oxide and reactive oxygen species and inducing apoptosis in LdDsRed-infected macrophages. Thus, these observations established the applicability of LdDsRed transfectants for flow cytometry-based antileishmanial screening. Further efforts aimed at establishing a high-throughput screening assay and determining the in vivo screening of potential antileishmanial leads are required.

  8. Enhanced efflux of (/sup 3/H)vinblastine from Chinese hamster ovary cells transfected with a full-length complementary DNA clone for the mdr1 gene

    SciTech Connect

    Hammond, J.R.; Johnstone, R.M.; Gros, P.

    1989-07-15

    Multidrug-resistant Chinese hamster ovary cell clones stably transfected with, and overexpressing, the mouse mdr1 complementary DNA clone along with drug-sensitive Chinese hamster ovary control cells were characterized for their capacities to accumulate and retain (/sup 3/H)vinblastine. Multidrug-resistant mdr1 transfectants show a 3-4-fold decrease in (/sup 3/H)vinblastine accumulation, compared to their drug-sensitive counterparts. After ATP depletion, this difference in (/sup 3/H)vinblastine accumulation between mdr1 transfectants and control cells effectively disappears. This ATP-dependent decreased drug accumulation is paralleled in mdr1 transfectants by an enhanced capacity of these cells to extrude the drug in an ATP-dependent manner. In medium containing glucose and glutamine, the mdr1 transfectants release preloaded drug at a rate five times that of control, drug-sensitive cells. In ATP-depleted control and mdr1-transfected cells, there is little difference in the rate or extent of (/sup 3/H)vinblastine release. The observation that the mdr1 transfectants show a decreased (/sup 3/H)vinblastine accumulation and an increased vinblastine release, both of which are abolished when cellular ATP levels are reduced, provides a direct demonstration that the product of the transfected mdr1 gene is responsible for a mechanism controlling cellular drug levels in an ATP-dependent manner. However, attempts to establish competition for (/sup 3/H)vinblastine transport by vincristine, daunomycin, and actinomycin D were only partly successful in mdr1 transfectants.

  9. Bioluminescence: a versatile technique for imaging cellular and molecular features

    PubMed Central

    Paley, Miranda A.

    2016-01-01

    Bioluminescence is a ubiquitous imaging modality for visualizing biological processes in vivo. This technique employs visible light and interfaces readily with most cell and tissue types, making it a versatile technology for preclinical studies. Here we review basic bioluminescence imaging principles, along with applications of the technology that are relevant to the medicinal chemistry community. These include noninvasive cell tracking experiments, analyses of protein function, and methods to visualize small molecule metabolites. In each section, we also discuss how bioluminescent tools have revealed insights into experimental therapies and aided drug discovery. Last, we highlight the development of new bioluminescent tools that will enable more sensitive and multi-component imaging experiments and, thus, expand our broader understanding of living systems. PMID:27594981

  10. Bioluminescence for USP sterility testing of pharmaceutical suspension products.

    PubMed Central

    Bussey, D M; Tsuji, K

    1986-01-01

    Bioluminescence measurement significantly improved the accuracy, sensitivity, precision, and reliability of the current visual endpoint determination for the USP sterility test and eliminated the day 7 transfer/dilution step required for testing suspension products. Thirteen strains of bacteria and fungi (representing potential contaminants in sterile products), three pharmaceutical suspension products, and four media were used in the experiment. No interference from suspension products was encountered in the detection of microbial growth by the bioluminescence measurement. The poor fungal growth encountered was attributed to insufficient diffusion of oxygen into the medium and was circumvented by use of a large tube size (38 by 200 mm) or by vortexing the medium once during the 2-week incubation period. Bioluminescence measurement would facilitate automated handling of the sterility test endpoint readout operation. The optimum parameters of bioluminescence measurement for application in sterility testing were determined. PMID:3954348

  11. Bioluminescence-Activated Deep-Tissue Photodynamic Therapy of Cancer

    PubMed Central

    Kim, Yi Rang; Kim, Seonghoon; Choi, Jin Woo; Choi, Sung Yong; Lee, Sang-Hee; Kim, Homin; Hahn, Sei Kwang; Koh, Gou Young; Yun, Seok Hyun

    2015-01-01

    Optical energy can trigger a variety of photochemical processes useful for therapies. Owing to the shallow penetration of light in tissues, however, the clinical applications of light-activated therapies have been limited. Bioluminescence resonant energy transfer (BRET) may provide a new way of inducing photochemical activation. Here, we show that efficient bioluminescence energy-induced photodynamic therapy (PDT) of macroscopic tumors and metastases in deep tissue. For monolayer cell culture in vitro incubated with Chlorin e6, BRET energy of about 1 nJ per cell generated as strong cytotoxicity as red laser light irradiation at 2.2 mW/cm2 for 180 s. Regional delivery of bioluminescence agents via draining lymphatic vessels killed tumor cells spread to the sentinel and secondary lymph nodes, reduced distant metastases in the lung and improved animal survival. Our results show the promising potential of novel bioluminescence-activated PDT. PMID:26000054

  12. Bioluminescence-activated deep-tissue photodynamic therapy of cancer.

    PubMed

    Kim, Yi Rang; Kim, Seonghoon; Choi, Jin Woo; Choi, Sung Yong; Lee, Sang-Hee; Kim, Homin; Hahn, Sei Kwang; Koh, Gou Young; Yun, Seok Hyun

    2015-01-01

    Optical energy can trigger a variety of photochemical processes useful for therapies. Owing to the shallow penetration of light in tissues, however, the clinical applications of light-activated therapies have been limited. Bioluminescence resonant energy transfer (BRET) may provide a new way of inducing photochemical activation. Here, we show that efficient bioluminescence energy-induced photodynamic therapy (PDT) of macroscopic tumors and metastases in deep tissue. For monolayer cell culture in vitro incubated with Chlorin e6, BRET energy of about 1 nJ per cell generated as strong cytotoxicity as red laser light irradiation at 2.2 mW/cm(2) for 180 s. Regional delivery of bioluminescence agents via draining lymphatic vessels killed tumor cells spread to the sentinel and secondary lymph nodes, reduced distant metastases in the lung and improved animal survival. Our results show the promising potential of novel bioluminescence-activated PDT.

  13. Selected Least Studied but not Forgotten Bioluminescent Systems.

    PubMed

    Oba, Yuichi; Stevani, Cassius V; Oliveira, Anderson G; Tsarkova, Aleksandra S; Chepurnykh, Tatiana V; Yampolsky, Ilia V

    2017-03-01

    Bioluminescence is a form of chemiluminescence generated by luminous organisms. Luminous taxa have currently been reported from about 800 genera and probably over 10 000 species in the world. On the other hand, their bioluminescent systems, including chemical structures of luciferins/chromophores and the genes encoding luciferases/photoproteins, have been elucidated from only a few taxonomic groups, for example beetles, bacteria, dinoflagellates, ostracods and some cnidarians. Research efforts to understand unknown bioluminescence systems are being conducted around the world, and recently, for example, novel luciferin structures of luminous enchytraeid potworms and fungi were identified by the authors. In this study, we review the current status and perspectives, in the context of postgenomic era, of most likely novel but less-revealed bioluminescence systems of ten selected organisms: earthworm, parchment tubeworm, fireworm, scaleworm, limpet, millipede, brittle star, acorn worms, tunicate and shark, which indeed are the next focus of our international collaboration.

  14. Digital spectral separation methods and systems for bioluminescence imaging.

    PubMed

    Wang, Ge; Shen, Haiou; Liu, Ying; Cong, Alex; Cong, Wenxiang; Wang, Yue; Dubey, Purnima

    2008-02-04

    We propose a digital spectral separation (DSS) system and methods to extract spectral information optimally from a weak multi-spectral signal such as in the bioluminescent imaging (BLI) studies. This system utilizes our newly invented spatially-translated spectral-image mixer (SSM), which consists of dichroic beam splitters, a mirror, and a DSS algorithm. The DSS approach overcomes the shortcomings of the data acquisition scheme used for the current BLI systems. Primarily, using our DSS scheme, spectral information will not be filtered out. Accordingly, truly parallel multi-spectral multi-view acquisition is enabled for the first time to minimize experimental time and optimize data quality. This approach also permits recovery of the bioluminescent signal time course, which is useful to study the kinetics of multiple bioluminescent probes using multi-spectral bioluminescence tomography (MSBT).

  15. Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication.

    PubMed Central

    Ladner, S K; Otto, M J; Barker, C S; Zaifert, K; Wang, G H; Guo, J T; Seeger, C; King, R W

    1997-01-01

    We report the development and isolation of a cell line, termed HepAD38, that replicates human hepatitis B virus (HBV) under conditions that can be regulated with tetracycline. In the presence of the antibiotic, this cell line is free of virus due to the repression of pregenomic (pg) RNA synthesis. Upon removal of tetracycline from the culture medium, the cells express viral pg RNA, accumulate subviral particles in the cytoplasm that contain DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should be useful for analyses of the viral replication cycle that depend upon viral DNA synthesis in a synchronized fashion. In addition, this cell line has been formatted into a high-throughput, cell-based assay that permits the large-scale screening of diverse compound libraries for new classes of inhibitors of HBV replication. PMID:9257747

  16. Biological water quality monitoring using chemiluminescent and bioluminescent techniques

    NASA Technical Reports Server (NTRS)

    Thomas, R. R.

    1978-01-01

    Automated chemiluminescence and bioluminescence sensors were developed for the continuous monitoring of microbial levels in water supplies. The optimal chemical procedures were determined for the chemiluminescence system to achieve maximum sensitivity. By using hydrogen peroxide, reaction rate differentiation, ethylene diamine tetraacetic acid (EDTA), and carbon monoxide pretreatments, factors which cause interference were eliminated and specificity of the reaction for living and dead bacteria was greatly increased. By employing existing technology with some modifications, a sensitive and specific bioluminescent system was developed.

  17. Toward Contactless Biology: Acoustophoretic DNA Transfection

    PubMed Central

    Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

    2016-01-01

    Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors. PMID:26828312

  18. Toward Contactless Biology: Acoustophoretic DNA Transfection

    NASA Astrophysics Data System (ADS)

    Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

    2016-02-01

    Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors.

  19. Basic and Applied Aspects of Color Tuning of Bioluminescence Systems

    NASA Astrophysics Data System (ADS)

    Ohmiya, Yoshihiro

    2005-09-01

    V. Viviani et al. [Biochemistry 38 (1999) 8271] were the first to succeed in cloning the red-emitting enzyme from the South American railroad worm, which is the only bioluminescent organism known to emit a red-colored light. The application of red bioluminescence has been our goal because the transmittance of longer-wavelength light is superior to that of the other colors for visualization of biological functions in living cells. Now, different color luciferases, which emit with wavelength maxima ranging from 400 to 630 nm, are available and are being used. For example, based on different color luciferases, Nakajima et al. developed a tricolor reporter in vitro assay system based on these different color luciferases in which the expression of three genes can be monitored simultaneously. On the other hand, bioluminescence resonance energy transfer (BRET) is a natural phenomenon caused by the intermolecular interaction between a bioluminescent protein and a fluorophore on a second protein, resulting in the light from the bioluminescence reaction having the spectrum of the fluorophore. Otsuji et al. [Anal. Biochem. 329 (2004) 230] showed that the change in the efficiency of energy transfer in intramolecular BRET can quantify cellular functions in living cells. In this review, I introduce the basic mechanisms of color tuning in bioluminescent systems and new applications based on color tuning in the life sciences.

  20. Bioluminescence imaging of Chlamydia muridarum ascending infection in mice.

    PubMed

    Campbell, Jessica; Huang, Yumeng; Liu, Yuanjun; Schenken, Robert; Arulanandam, Bernard; Zhong, Guangming

    2014-01-01

    Chlamydial pathogenicity in the upper genital tract relies on chlamydial ascending from the lower genital tract. To monitor chlamydial ascension, we engineered a luciferase-expressing C. muridarum. In cells infected with the luciferase-expressing C. muridarum, luciferase gene expression and enzymatic activity (measured as bioluminescence intensity) correlated well along the infection course, suggesting that bioluminescence can be used for monitoring chlamydial replication. Following an intravaginal inoculation with the luciferase-expressing C. muridarum, 8 of 10 mice displayed bioluminescence signal in the lower with 4 also in the upper genital tracts on day 3 after infection. By day 7, all 10 mice developed bioluminescence signal in the upper genital tracts. The bioluminescence signal was maintained in the upper genital tract in 6 and 2 mice by days 14 and 21, respectively. The bioluminescence signal was no longer detectable in any of the mice by day 28. The whole body imaging approach also revealed an unexpected airway infection following the intravaginal inoculation. Although the concomitant airway infection was transient and did not significantly alter the genital tract infection time courses, caution should be taken during data interpretation. The above observations have demonstrated that C. muridarum can not only achieve rapid ascending infection in the genital tract but also cause airway infection following a genital tract inoculation. These findings have laid a foundation for further optimizing the C. muridarum intravaginal infection murine model for understanding chlamydial pathogenic mechanisms.

  1. Stimulation of bioluminescence in Noctiluca sp. using controlled temperature changes.

    PubMed

    Han, Jing; Li, GuiJuan; Liu, HuanYing; Hu, HaoHao; Zhang, XueGang

    2013-01-01

    Bioluminescence induced by multifarious stimuli has long been observed and is remains under investigation because of its great complexity. In particular, the exact mechanism underlying bioluminescence is not yet fully understood. This work presents a new experimental method for studying Noctiluca sp. bioluminescence under temperature change stimulation. It is a study of Noctiluca sp. bioluminescence using controlled temperature changes in a tank. A characteristic of this experiment is the large volume of water used (1 m(3) in a tank of 2 × 1 × 1 m). Temperature changes were controlled by two methods. In the first, a flask filled with hot water was introduced into the tank and in the second, a water heater was used in the tank. Temperature changes were recorded using sensors. Noctiluca sp. bioluminescence was recorded using a Canon 5D Mark II and this allowed the characteristics of Noctiluca sp. bioluminescence under temperature change stimulation to be monitored.

  2. 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.

  3. High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial Lux reporter.

    PubMed

    Class, Bradley; Thorne, Natasha; Aguisanda, Francis; Southall, Noel; McKew, John C; Zheng, Wei

    2015-04-01

    Cell viability assays are extensively used to determine cell health, evaluate growth conditions, and assess compound cytotoxicity. Most existing assays are endpoint assays, in which data are collected at one time point after termination of the experiment. The time point at which toxicity of a compound is evident, however, depends on the mechanism of that compound. An ideal cell viability assay allows the determination of compound toxicity kinetically without having to terminate the assay prematurely. We optimized and validated a reagent-addition-free cell viability assay using an autoluminescent HEK293 cell line that stably expresses bacterial luciferase and all substrates necessary for bioluminescence. This cell viability assay can be used for real-time, long-term measurement of compound cytotoxicity in live cells with a signal-to-basal ratio of 20- to 200-fold and Z-factors of ~0.6 after 24-, 48- 72-, or 96-h incubation with compound. We also found that the potencies of nine cytotoxic compounds correlated well with those measured by four other commonly used cell viability assays. The results demonstrated that this kinetic cell viability assay using the HEK293(lux) autoluminescent cell line is useful for high-throughput evaluation of compound cytotoxicity.

  4. High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.

    PubMed

    Ausseil, Frederic; Samson, Arnaud; Aussagues, Yannick; Vandenberghe, Isabelle; Creancier, Laurent; Pouny, Isabelle; Kruczynski, Anna; Massiot, Georges; Bailly, Christian

    2007-02-01

    To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.

  5. Stable transfection and identification of a hair follicle-specific expression vector of IGFBP-5 in goat fetal fibroblasts.

    PubMed

    Wang, X J; Su, H M; Liang, Y; Wang, Y F; Guo, X D; Wang, Z G; Liu, D J

    2014-03-17

    The insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the 6 members of the IGFBP family and is involved in the regulation of cell growth, apoptosis, and other IGF-stimulated signaling pathways. To determine the significance of IGFBP-5 in the Inner Mongolia Cashmere goat (Capra hircus), a hair follicle-specific expression vector of IGFBP-5, pCDsRed2-K-IGFBP5 (6.7 kb), was constructed by cloning IGFBP-5 downstream of the keratin-association protein (KAP)6-1 promoter and inserting this fragment into pCDsRed2, which contains a red fluorescent protein (DsRed) expression unit. Inner Mongolia Cashmere goat fetal fibroblast (GFb) cells were transfected with the expression vector by using Lipofectamine(TM) 2000. Cell clones that stably expressed red fluorescence were obtained after selection with Geneticin (G418). The transgene in the cell clones was examined by polymerase chain reaction to verify that exogenous DNA (pKAP6-1 and IGFBP-5) had integrated stably into GFb cells. These data suggest that this method can be used for the construction of a hair follicle-specific expression vector for functional genetic analyses and for obtaining stable transfection donor cells for nuclear transfer.

  6. Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques

    PubMed Central

    2011-01-01

    Background Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun") delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI) methods. Results Plasmid DNA carrying the firefly luciferase (LUC) reporter gene under the control of the human Cytomegalovirus (CMV) promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter) using different DNA Loading Ratios (DLRs), and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50) at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. Conclusions The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results demonstrate that

  7. A General RNA Motif for Cellular Transfection

    PubMed Central

    Magalhães, Maria LB; Byrom, Michelle; Yan, Amy; Kelly, Linsley; Li, Na; Furtado, Raquel; Palliser, Deborah; Ellington, Andrew D; Levy, Matthew

    2012-01-01

    We have developed a selection scheme to generate nucleic acid sequences that recognize and directly internalize into mammalian cells without the aid of conventional delivery methods. To demonstrate the generality of the technology, two independent selections with different starting pools were performed against distinct target cells. Each selection yielded a single highly functional sequence, both of which folded into a common core structure. This internalization signal can be adapted for use as a general purpose reagent for transfection into a wide variety of cell types including primary cells. PMID:22233578

  8. Bioluminescence tracking of alginate micro-encapsulated cell transplants.

    PubMed

    Tiernan, Aubrey R; Sambanis, Athanassios

    2017-02-01

    Cell-based therapies to treat loss-of-function hormonal disorders such as diabetes and Parkinson's disease are routinely coupled with encapsulation strategies, but an understanding of when and why grafts fail in vivo is lacking. Consequently, investigators cannot clearly define the key factors that influence graft success. Although bioluminescence is a popular method to track the survival of free cells transplanted in preclinical models, little is known of the ability to use bioluminescence for real-time tracking of microencapsulated cells. Furthermore, the impact that dynamic imaging distances may have, due to freely-floating microcapsules in vivo, on cell survival monitoring is unknown. This work addresses these questions by applying bioluminescence to a pancreatic substitute based on microencapsulated cells. Recombinant insulin-secreting cells were transduced with a luciferase lentivirus and microencapsulated in Ba(2+) crosslinked alginate for in vitro and in vivo studies. In vitro quantitative bioluminescence monitoring was possible and viable microencapsulated cells were followed in real time under both normoxic and anoxic conditions. Although in vivo dispersion of freely-floating microcapsules in the peritoneal cavity limited the analysis to a qualitative bioluminescence evaluation, signals consistently four orders of magnitude above background were clear indicators of temporal cell survival. Strong agreement between in vivo and in vitro cell proliferation over time was discovered by making direct bioluminescence comparisons between explanted microcapsules and parallel in vitro cultures. Broader application of this bioluminescence approach to retrievable transplants, in supplement to currently used end-point physiological tests, could improve understanding and accelerate development of cell-based therapies for critical clinical applications. Copyright © 2014 John Wiley & Sons, Ltd.

  9. Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters.

    PubMed

    Ramanathan, Chidambaram; Khan, Sanjoy K; Kathale, Nimish D; Xu, Haiyan; Liu, Andrew C

    2012-09-27

    In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere. Individual cells are the functional units for generation and maintenance of circadian rhythms, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms. Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection or stable transduction. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host

  10. Isolation, culture, and transfection of melanocytes.

    PubMed

    Godwin, Lauren S; Castle, Joanna T; Kohli, Jaskaren S; Goff, Philip S; Cairney, Claire J; Keith, W Nicol; Sviderskaya, Elena V; Bennett, Dorothy C

    2014-06-03

    Located in the basal epidermis and hair follicles, melanocytes of the integument are responsible for its coloration through production of melanin pigments. Melanin is produced in lysosomal-like organelles called melanosomes. In humans, this skin pigmentation acts as an ultraviolet radiation filter. Abnormalities in the division of melanocytes are quite common, with potentially oncogenic growth usually followed by cell senescence producing benign naevi (moles), or occasionally melanoma. Therefore, melanocytes are a useful model for studying melanoma, as well as pigmentation and organelle transport and the diseases affecting these mechanisms. This chapter focuses on the isolation, culture, and transfection of human and murine melanocytes. The first basic protocol describes the primary culture of melanocytes from human skin and the maintenance of growing cultures. The second basic protocol details the subculture and preparation of mouse keratinocyte feeder cells. The primary culture of melanocytes from mouse skin is described in the third basic protocol, and, lastly, the fourth basic protocol outlines a technique for transfecting melanocytes and melanoma cells.

  11. Optimization of conditions for transfection with the Sofast gene vector.

    PubMed

    Zhou, Lei; Liu, Fan; Qiao, Fang-Fang; Tong, Man-Li; Fu, Zuo-Gen; Dan, Bing; Yang, Tian-Ci; Zhang, Zhong-Ying

    2011-01-01

    We previously reported the synthesis and characterization of a novel cationic polymer gene vector. The present article further explored and optimized the working conditions of the Sofast gene vector both in vitro and in vivo, and improved its performance. The transfection conditions of Sofast, such as cell type, cell density, transfection time, N/P values and analysis time after transfection, were further explored. Moreover, the effects of the fusion peptide diINF-7 on transfection efficiency were examined. Sofast was successfully applied for the transfection of exogenous genes into more than 40 types of cell lines derived from humans, mice, monkeys and other species. When the cells were 50-80% confluent, Sofast possessed a better transfection efficiency. In most cases, Sofast also had a higher transfection efficiency when it was used to transfect cells that were seeded for several hours and had adhered to the substrate. The results from in vitro experiments indicate that the recommended Sofast to DNA mass ratio is 16:1, and the optimum analysis time after transfection is 48 h. The salt concentration in the Sofast working solution markedly affected the transfection efficiency. When conducting in vivo transfection, the working solution should be salt-free, whereas for in vitro transfection, it is more appropriate for the working solution to include certain salt concentrations. Finally, the results confirm that diINF-7 significantly promotes the transfection efficiency of Sofast. In conclusion, the present research not only established the optimal conditions for Sofast in the transfection of commonly used cells, but also built the foundations for in vivo and in vitro applications of Sofast, as well as its use in clinical practice.

  12. Thoughts on the diversity of convergent evolution of bioluminescence on earth

    NASA Astrophysics Data System (ADS)

    Waldenmaier, Hans E.; Oliveira, Anderson G.; Stevani, Cassius V.

    2012-10-01

    The widespread independent evolution of analogous bioluminescent systems is one of the most impressive and diverse examples of convergent evolution on earth. There are roughly 30 extant bioluminescent systems that have evolved independently on Earth, with each system likely having unique enzymes responsible for catalysing the bioluminescent reaction. Bioluminescence is a chemical reaction involving a luciferin molecule and a luciferase or photoprotein that results in the emission of light. Some independent systems utilize the same luciferin, such as the use of tetrapyrrolic compounds by krill and dinoflagellates, and the wide use of coelenterazine by marine organisms, while the enzymes involved are unique. One common thread among all the different bioluminescent systems is the requirement of molecular oxygen. Bioluminescence is found in most forms of life, especially marine organisms. Bioluminescence in known to benefit the organism by: attraction, repulsion, communication, camouflage, and illumination. The marine ecosystem is significantly affected by bioluminescence, the only light found in the pelagic zone and below is from bioluminescent organisms. Transgenic bioluminescent organisms have revolutionized molecular research, medicine and the biotechnology industry. The use of bioluminescence in studying molecular pathways and disease allows for non-invasive and real-time analysis. Bioluminescence-based assays have been developed for several analytes by coupling luminescence to many enzyme-catalysed reactions.

  13. Effect of halogenated fluorescent compounds on bioluminescent reactions.

    PubMed

    Kirillova, Tamara N; Gerasimova, Marina A; Nemtseva, Elena V; Kudryasheva, Nadezhda S

    2011-04-01

    The paper investigates an application of luminescent bioassays to monitor the toxicity of organic halides. Effects of xanthene dyes (fluorescein, eosin Y, and erythrosin B), used as model compounds, on bioluminescent reactions of firefly Luciola mingrelica, marine bacteria Photobacterium leiognathi, and hydroid polyp Obelia longissima were studied. Dependence of bioluminescence quenching constants on the atomic weight of halogen substituents in dye molecules was demonstrated. Bacterial bioluminescence was shown to be most sensitive to heavy halogen atoms involved in molecular structure; hence, it is suitable for construction of sensors to monitor toxicity of halogenated compounds. Mechanisms of bioluminescence quenching--energy transfer processes, collisional interactions, and enzyme-dye binding--were considered. Changes of bioluminescence (BL) spectra in the presence of the dyes were analyzed. Interactions of the dyes with enzymes were studied using fluorescence characteristics of the dyes in steady-state and time-resolved experiments. The dependences of fluorescence anisotropy of enzyme-bound dyes, the average fluorescence lifetime, and the number of exponential components in fluorescence decay on the atomic weight of halogen substituents were demonstrated. The results are discussed in terms of "dark effect of heavy halogen atom" in the process of enzyme-dye binding; hydrophobic interactions were assumed to be responsible for the effect.

  14. Spectrally resolved bioluminescence tomography using the reciprocity approach

    PubMed Central

    Dehghani, Hamid; Davis, Scott C.; Pogue, Brian W.

    2008-01-01

    Spectrally resolved bioluminescence optical tomography is an approach to recover images of, for example, Luciferase activity within a volume using multiwavelength emission data from internal bioluminescence sources. The underlying problem of uniqueness associated with nonspectrally resolved intensity-based bioluminescence tomography is demonstrated and it is shown that using a non-negative constraint inverse algorithm, an accurate solution for the source distribution can be calculated from the measured data. Reconstructed images of bioluminescence are presented using both simulated complex and heterogeneous small animal models as well as real multiwavelength data from a tissue-simulating phantom. The location of the internal bioluminescence source using experimental data is obtained with 0.5 mm accuracy and it is shown that small (2.5 mm diameter) sources of up to 12.5 mm deep, within a complex mouse model, can be resolved accurately using a single view data collection strategy. Finally, using the reciprocity approach for image reconstruction, a dramatic improvement in computational time is shown without loss to image accuracy with both experimental and simulated data, potentially reducing computing time from 402 to 3.75 h. PMID:19070220

  15. Detection of DNA adducts by bioluminescence

    NASA Astrophysics Data System (ADS)

    Xu, Shunqing; Tan, Xianglin; Yao, Qunfeng; He, Min; Zhou, Yikai; Chen, Jian

    2001-09-01

    Luminescent assay for detection ATP is very sensitive with limitation of 10-17 moles. ATP using styrene oxide as a model carcinogen we currently apply a luminescence technique to detect the very low levels of carcinogen-DNA adducts in vitro and in vivo. The bioluminescent assay of DNA adducts entails three consecutive steps: digestion of modified DNA to adducted dinucleoside monophosphate and normal nucleotide are hydrolyzed to nucleosides (N) by nuclease P1 and prostatic acid phosphomonesterase (PAP); incorporation of (gamma) -P of ATP into normal nucleoside(N); detection of consumption of ATP by luminescence. This assay does not require separate manipulation because of the selective property of nuclease P1. One fmol of carcinogen- DNA adducts was detected by luminescent assay. A good correlation between results of luminescent assay and 32P-postlabeling procedures has been observed. We detect 1 adduct in 108 nucleotides for 10(mu) g DNA sample. The procedures of luminescent method is very simple and low- cost. IT appears applicable to the ultra sensitive detection of low levels of DNA adducts without radioactive isotope.

  16. Enhancement of the efficiency of femtosecond optical transfection

    NASA Astrophysics Data System (ADS)

    Praveen, Bavishna B.; Stevenson, David; Antkowiak, Maciej; Gunn-Moore, Frank J.; Dholakia, Kishan

    2010-12-01

    Cell transfection is the process in which extra cellular nucleic acids such as DNA, RNA, Si-RNA can be deliberately injected into the cytoplasm of the cell. This technique of cell transfection forms a central tool in the hands of a cell biologist to explore the mechanism within the cell. In optical transfection a well focused laser spot alters the permeability of the cell membrane so as to allow the entry of extra-nuclear materials into the cell. Femto-second optical transfection have proved to be better than other laser based cell transfection, owing to the three dimensionally confined multi-photon effects on the cell membrane thereby leaving the rest of the cell unaffected. Even though the femto-second optical transfection has proved to be sterile, non-invasive and highly selective, it has to improve in terms of efficiency, and throughput to address real life problems. We report here a method to achieve significant enhancement in the efficiency of femto-second optical transfection. The protocol of the transfection procedure is modified by adding a suitable biochemical reagent - Nupherin-neuron - into the cell medium during the transfection, which can assist the delivery of DNA into the nucleus once the DNA gets injected into the cytoplasm of the cell. We achieved a 3 fold enhancement in the transfection efficiency with this modified protocol. Also we report for the first time the transfection of recently trypsinised cells with a very high transfection efficiency, which would pave way to the development of high throughput microfluidic optical transfection devices.

  17. Enhancement of the efficiency of femtosecond optical transfection

    NASA Astrophysics Data System (ADS)

    Praveen, Bavishna B.; Stevenson, David; Antkowiak, Maciej; Gunn-Moore, Frank J.; Dholakia, Kishan

    2011-08-01

    Cell transfection is the process in which extra cellular nucleic acids such as DNA, RNA, Si-RNA can be deliberately injected into the cytoplasm of the cell. This technique of cell transfection forms a central tool in the hands of a cell biologist to explore the mechanism within the cell. In optical transfection a well focused laser spot alters the permeability of the cell membrane so as to allow the entry of extra-nuclear materials into the cell. Femto-second optical transfection have proved to be better than other laser based cell transfection, owing to the three dimensionally confined multi-photon effects on the cell membrane thereby leaving the rest of the cell unaffected. Even though the femto-second optical transfection has proved to be sterile, non-invasive and highly selective, it has to improve in terms of efficiency, and throughput to address real life problems. We report here a method to achieve significant enhancement in the efficiency of femto-second optical transfection. The protocol of the transfection procedure is modified by adding a suitable biochemical reagent - Nupherin-neuron - into the cell medium during the transfection, which can assist the delivery of DNA into the nucleus once the DNA gets injected into the cytoplasm of the cell. We achieved a 3 fold enhancement in the transfection efficiency with this modified protocol. Also we report for the first time the transfection of recently trypsinised cells with a very high transfection efficiency, which would pave way to the development of high throughput microfluidic optical transfection devices.

  18. Bacterial bioluminescence as a lure for marine zooplankton and fish.

    PubMed

    Zarubin, Margarita; Belkin, Shimshon; Ionescu, Michael; Genin, Amatzia

    2012-01-17

    The benefits of bioluminescence for nonsymbiotic marine bacteria have not been elucidated fully. One of the most commonly cited explanations, proposed more than 30 y ago, is that bioluminescence augments the propagation and dispersal of bacteria by attracting fish to consume the luminous material. This hypothesis, based mostly on the prevalence of luminous bacteria in fish guts, has not been tested experimentally. Here we show that zooplankton that contacts and feeds on the luminescent bacterium Photobacterium leiognathi starts to glow, and demonstrate by video recordings that glowing individuals are highly vulnerable to predation by nocturnal fish. Glowing bacteria thereby are transferred to the nutritious guts of fish and zooplankton, where they survive digestion and gain effective means for growth and dispersal. Using bioluminescence as bait appears to be highly beneficial for marine bacteria, especially in food-deprived environments of the deep sea.

  19. Bacterial bioluminescence as a lure for marine zooplankton and fish

    PubMed Central

    Zarubin, Margarita; Belkin, Shimshon; Ionescu, Michael; Genin, Amatzia

    2012-01-01

    The benefits of bioluminescence for nonsymbiotic marine bacteria have not been elucidated fully. One of the most commonly cited explanations, proposed more than 30 y ago, is that bioluminescence augments the propagation and dispersal of bacteria by attracting fish to consume the luminous material. This hypothesis, based mostly on the prevalence of luminous bacteria in fish guts, has not been tested experimentally. Here we show that zooplankton that contacts and feeds on the luminescent bacterium Photobacterium leiognathi starts to glow, and demonstrate by video recordings that glowing individuals are highly vulnerable to predation by nocturnal fish. Glowing bacteria thereby are transferred to the nutritious guts of fish and zooplankton, where they survive digestion and gain effective means for growth and dispersal. Using bioluminescence as bait appears to be highly beneficial for marine bacteria, especially in food-deprived environments of the deep sea. PMID:22203999

  20. The Expanding Toolbox of In Vivo Bioluminescent Imaging

    PubMed Central

    Xu, Tingting; Close, Dan; Handagama, Winode; Marr, Enolia; Sayler, Gary; Ripp, Steven

    2016-01-01

    In vivo bioluminescent imaging (BLI) permits the visualization of engineered bioluminescence from living cells and tissues to provide a unique perspective toward the understanding of biological processes as they occur within the framework of an authentic in vivo environment. The toolbox of in vivo BLI includes an inventory of luciferase compounds capable of generating bioluminescent light signals along with sophisticated and powerful instrumentation designed to detect and quantify these light signals non-invasively as they emit from the living subject. The information acquired reveals the dynamics of a wide range of biological functions that play key roles in the physiological and pathological control of disease and its therapeutic management. This mini review provides an overview of the tools and applications central to the evolution of in vivo BLI as a core technology in the preclinical imaging disciplines. PMID:27446798

  1. Leishmania tropica experimental infection in the rat using luciferase-transfected parasites.

    PubMed

    Talmi-Frank, Dalit; Jaffe, Charles L; Nasereddin, Abedelmajeed; Baneth, Gad

    2012-06-08

    Leishmania tropica is the causative agent of zoonotic cutaneous leishmaniasis in different parts of the Old World. Although it is a common cause of disease in some areas of the world, there is insufficient knowledge on the pathogenicity of this parasite in mammalian hosts and animal models. L. tropica luciferase-transfected metacyclic-stage promastigotes were inoculated into the footpad or ear of Sprague Dawley (SD) rats. Parasite DNA was detected by kDNA real time PCR in the blood at varying levels from 2 days to 5 weeks post infection (PI) in the absence of clinical signs. Parasite DNA was found in the spleen of all rats at the end of the study, and the parasitic load was up to 40 times higher in the spleen when compared with inoculation sites. Parasites were cultured from the spleen, and skin inoculation sites 5 weeks PI. Bioluminescent parasites were observed by in vivo imaging at one day PI, but the technique was not sufficiently sensitive to follow parasite spread after this time. This study provides new evidence for the viscerotropic spread of L. tropica in the rat and demonstrates that the rat can serve as a model for persistent visceralizing infection with this parasite.

  2. In Vivo Bioluminescence Imaging for Longitudinal Monitoring of Inflammation in Animal Models of Uveitis

    PubMed Central

    Gutowski, Michal B.; Wilson, Leslie; Van Gelder, Russell N.; Pepple, Kathryn L.

    2017-01-01

    Purpose We develop a quantitative bioluminescence assay for in vivo longitudinal monitoring of inflammation in animal models of uveitis. Methods Three models of experimental uveitis were induced in C57BL/6 albino mice: primed mycobacterial uveitis (PMU), endotoxin-induced uveitis (EIU), and experimental autoimmune uveitis (EAU). Intraperitoneal injection of luminol sodium salt, which emits light when oxidized, provided the bioluminescence substrate. Bioluminescence images were captured by a PerkinElmer In Vivo Imaging System (IVIS) Spectrum and total bioluminescence was analyzed using Living Image software. Bioluminescence on day zero was compared to bioluminescence on the day of peak inflammation for each model. Longitudinal bioluminescence imaging was performed in EIU and EAU. Results In the presence of luminol, intraocular inflammation generates detectable bioluminescence in three mouse models of uveitis. Peak bioluminescence in inflamed PMU eyes (1.46 × 105 photons/second [p/s]) was significantly increased over baseline (1.47 × 104 p/s, P = 0.01). Peak bioluminescence in inflamed EIU eyes (3.18 × 104 p/s) also was significantly increased over baseline (1.09 × 104 p/s, P = 0.04), and returned to near baseline levels by 48 hours. In EAU, there was a nonsignificant increase in bioluminescence at peak inflammation. Conclusions In vivo bioluminescence may be used as a noninvasive, quantitative measure of intraocular inflammation in animal models of uveitis. Primed mycobacterial uveitis and EIU are both acute models with robust anterior inflammation and demonstrated significant changes in bioluminescence corresponding with peak inflammation. Experimental autoimmune uveitis is a more indolent posterior uveitis and generated a more modest bioluminescent signal. In vivo imaging system bioluminescence is a nonlethal, quantifiable assay that can be used for monitoring inflammation in animal models of uveitis. PMID:28278321

  3. Metal-enhanced bioluminescence: An approach for monitoring biological luminescent processes

    NASA Astrophysics Data System (ADS)

    Eltzov, Evgeni; Prilutsky, Daria; Kushmaro, Ariel; Marks, Robert S.; Geddes, Chris D.

    2009-02-01

    In this letter, the observation of metal (plasmon)-enhanced bioluminescence is reported. Bacteria, which are capable of generating specific bioluminescence signatures upon metabolic changes (general toxicity), have been studied from both glass and silvered glass microwell bottoms, where the silvered microwells have been modified with surface deposited silver island films (SiFs). The presence of the SiFs plasmon amplifies the near-field bioluminescence signatures, ≈<50 nm from the surface, enabling amplified detection of the reporter bioluminescence indicating sample toxicity. Using our approach a greater than fivefold enhancement in far-field bioluminescence occurs with much greater enhancements in the near-field predicted.

  4. [Expression of Photobacterium leiognathi bioluminescence system genes in Escherichia coli].

    PubMed

    Ptitsyn, L R; Fatova, M A; Stepanov, A I

    1990-02-01

    Expression of Photobacterium leiognathi bioluminescence genes under the control of lac, tac, tet promoters in Escherichia coli cells has been studied. The position of the genes for aliphatic aldehyde biosynthesis and for the synthesis of luciferase subunits was identified. The plasmid pBRPL1 has been constructed containing the system of bioluminescence genes devoid of promoter following the polylinker DNA fragment. The plasmid can be used for selection of promoter containing DNA sequences as well as for studying the promoters regulation in process of Escherichia coli cells growth.

  5. Aequorea victoria bioluminescence moves into an exciting new era.

    PubMed

    Kendall, J M; Badminton, M N

    1998-05-01

    Bioluminescence has revolutionized research into many cellular and molecular-biological processes, ranging from intracellular signalling to gene transcription. This article focuses on the chemistry and biotechnological exploitation of the two proteins involved in bioluminescence of the jellyfish Aequorea victoria--aequorin and green fluorescent protein. Engineered recombinant aequorin has led to a novel technological approach to monitoring calcium signals in organelles and subcellular domains. A new generation of intracellular calcium indicators has been produced in which engineered variants of green fluorescent protein are used to probe their ionic environment using intramolecular fluorescence-resonance-energy transfer.

  6. CCD imaging of basal bioluminescence in larval fireflies: clues on the anatomic origin and evolution of bioluminescence.

    PubMed

    Viviani, V R; Okawachi, F M; Scorsato, V; Abdalla, F C

    2008-04-01

    The anatomic and biochemical origin of beetle bioluminescence is still poorly understood. Through CCD imaging we report that larvae and pupae of the Brazilian fireflies Aspisoma lineatum and Cratomorphus sp emit a continuous weak glow throughout the entire body during all stages. This luminescence is especially developed after feeding, ecdysis and in the pupal stage, gradually disappearing as the cuticle becomes sclerotized and the adult emerges. This weak glow arises from the fat body, which consists of small lobes spread all over the body cavity. According to their pigmentation, these lobes can be divided in whitish and pinkish, and display different luciferase isozymes. Morphological studies suggest that the jelly-like ventral lanterns in the 8th abdominal segment evolved from these white lobes, providing a rationale for the widespread location of lanterns in larvae of different bioluminescent beetles. The biological and biochemical function of this weak diffuse bioluminescence is discussed in the context of the larval life-history.

  7. Specific growth stimulation by linoleic acid in hepatoma cell lines transfected with the target protein of a liver carcinogen.

    PubMed Central

    Keler, T; Barker, C S; Sorof, S

    1992-01-01

    The hepatic carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) was shown previously to interact specifically with its target protein, liver fatty acid binding protein (L-FABP), early during hepatocarcinogenesis in rats. In search of the significance of the interaction, rat L-FABP cDNA in the sense and antisense orientations was transfected into a subline of the rat hepatoma HTC cell line that did not express L-FABP. After the transfections, the basal doubling times of the cells were not significantly different. However, at 10(-5)-10(-7) M, linoleic acid, which is an essential fatty acid, a ligand of L-FABP, and the precursor of many eicosanoids and related lipids, stimulated the incorporation of [3H]thymidine in three randomly isolated and stably transfected cell clones that expressed L-FABP, but virtually did not stimulate the incorporation of [3H]thymidine in three L-FABP-nonexpressing clones transfected with the antisense DNA. Linoleic acid at 10(-6) M increased cell number almost 3-fold (38% vs. 14%; P less than 0.0001) and thymidine incorporation nearly 5-fold (23.2% vs. 4.9%; P less than 0.001) in the L-FABP-expressing cells compared to that in the transfected nonexpressing cells. L-FABP acted specifically and cooperatively with linoleic acid, inasmuch as all the proteins other than L-FABP in the transfected L-FABP nonexpressing cells and four other fatty acids (gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, and palmitoleic acid) were unable to effect a significant elevation or difference in the level of DNA synthesis that was attributable to the transfection. Metabolism of the linoleic acid to oxygenated derivatives was apparently necessary, since the cyclooxygenase inhibitor indomethacin partly inhibited and the antioxidant lipoxygenase inhibitors nordihydroguariaretic acid and alpha-tocopherol completely abolished the growth stimulation. The evidence supports the idea that L-FABP, the target protein of the liver carcinogen

  8. Transfection of mammalian cells using block copolypeptide vesicles.

    PubMed

    Sun, Victor Z; Choe, Uh-Joo; Rodriguez, April R; Dai, Howard; Deming, Timothy J; Kamei, Daniel T

    2013-05-01

    An arginine-leucine block copolypeptide (R60 L20 ) is synthesized, which is capable of forming vesicles with controllable sizes, able to transport hydrophilic cargo across the cell membrane, and exhibit relatively low cytotoxicity. The R60 L20 vesicles also possess the ability to deliver DNA into mammalian cells for transfection. Although the transfection efficiency is lower than that of the commercially available transfection agent Lipofectamine 2000, the R60 L20 vesicles are able to achieve transfection with significantly lower cytotoxicity and immunogenicity. This behavior is potentially due to its stronger interaction with DNA which subsequently provides better protection against anionic heparin.

  9. 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

  10. 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

    Krishnamoorthy, Archana; Robertson, J Brian

    2015-09-01

    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.

  11. A combination of NADHP and hispidin is not essential for bioluminescence in luminous fungal living gills of Mycena chlorophos.

    PubMed

    Teranishi, Katsunori

    2017-01-05

    The chemical mechanisms underlying visible bioluminescence in the fungus Mycena chlorophos are not clear. A combination of dihydronicotinamide adenine dinucleotide phosphate (NADPH) and hispidin, which has been reported to increase the intensity of in vitro luminescence in crude cold-water extracts prepared from the bioluminescent fruiting bodies of M. chlorophos, exhibited potential bioluminescence activation in the early bioluminescence stages, in which the bioluminescence was ultra-weak, for living gills and luminescence activation for non-bioluminescent gills, which was collapsed by freezing and subsequent thawing, at all bioluminescence stages. These abilities were not evident in considerably bioluminescent gills. These abilities were blocked by trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid, which were identified as in vivo bioluminescence-activating components. Original bioluminescence and bioluminescence produced from the addition of trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid in living gills were almost completely inhibited by 10 mM NaN3 , whereas the luminescence produced form the combination of NADPH and hispidin in thawed non-bioluminescent and living gills at the early weak bioluminescence stages was not inhibited by 10 mM NaN3 . Thus, the combination of NADPH and hispidin plays different roles in luminescence systems compared with essential bioluminescence systems, and the combination of NADPH and hispidin was not essential for visible bioluminescence in living gills.

  12. Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions.

    PubMed

    Yokawa, Satoru; Suzuki, Takahiro; Inouye, Satoshi; Inoh, Yoshikazu; Suzuki, Ryo; Kanamori, Takao; Furuno, Tadahide; Hirashima, Naohide

    2017-04-15

    We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion.

  13. Metabolic self-organization of bioluminescent Escherichia coli.

    PubMed

    Simkus, Remigijus; Baronas, Romas

    2011-01-01

    A possible reason for the complexity of the signals produced by bioluminescent biosensors might be self-organization of the cells. In order to verify this possibility, bioluminescence images of cultures of lux gene reporter Escherichia coli were recorded for several hours after being placed into 8-10 mm diameter cylindrical containers. It was found that luminous cells distribute near the three-phase contact line, forming irregular azimuthal waves. As we show, space-time plots of quasi-one-dimensional bioluminescence measured along the contact line can be simulated by reaction-diffusion-chemotaxis equations, in which the reaction term for the cells is a logistic (autocatalytic) growth function. It was found that the growth rate of the luminous cells (~0.02 s(-1)) is >100 times higher than the growth rate of E. coli. We provide an explanation for this result by assuming that E. coli exhibits considerable respiratory flexibility (the ability of oxygen-induced switching from one metabolic pathway to another). According to the simple two-state model presented here, the number of oxic (luminous) cells grows at the expense of anoxic (dark) cells, whereas the total number of (oxic and anoxic) cells remains unchanged. It is conjectured that the corresponding reaction-diffusion-chemotaxis model for bioluminescence pattern formation can be considered as a model for the energy-taxis and metabolic self-organization in the population of the metabolically flexible bacteria under hypoxic conditions.

  14. Dynamic Modeling of Marine Bioluminescence and Night Time Leaving Radiance

    DTIC Science & Technology

    2012-09-30

    physical model is based on the Navy Coastal Ocean Model (NCOM), the biochemical model simulates dynamics of two sizes of phytoplankton, zooplankton ...layer of bioluminescent zooplankton were replaced by water masses advected from the northern coast of the bay with a relatively high presence of mostly

  15. Leukocyte esterase-nitrite and bioluminescence assays as urine screens.

    PubMed Central

    Males, B M; Bartholomew, W R; Amsterdam, D

    1985-01-01

    The 1-min leukocyte esterase (LE)-nitrite test (Chemstrip 9; Biodynamics, Division of Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and a bioluminescence assay (Monolight centrifugation method; Analytical Luminescence Laboratory, Inc., San Diego, Calif.) were tested for their efficacy as urine screens among 453 patients at a tertiary-care teaching hospital. Both methods had the capacity to exclude significant bacteriuria (greater than or equal to 10(5) CFU/ml) when compared with the results of conventional culture methods, with predictive values of 99 and 93%, respectively, for a negative test. Bioluminescence was the more accurate nonculture method used. Sensitivity and specificity values were 97 and 71%, respectively, for bioluminescence, 82 and 60%, respectively, for LE with nitrite, and 72 and 64%, respectively, for LE without nitrite. At reduced levels of bacteriuria less than 10(5) CFU/ml), the sensitivities of LE-nitrite and bioluminescence were decreased but comparable. The addition of protein and blood test results in the Chemstrip 9, along with LE-nitrite as bacteriuria indicators, were unsatisfactory because of the large numbers of false-positive results attributed to protein and blood determinations. LE activity as detected by the LE test was a poor predictor of significant bacteriuria in both male and female patients. The sensitivity (71%) and specificity (57%) of the LE test in male patients were significantly lower than those previously reported and varied with the patient population studied. PMID:3935662

  16. Upgrading bioluminescent bacterial bioreporter performance by splitting the lux operon.

    PubMed

    Yagur-Kroll, Sharon; Belkin, Shimshon

    2011-05-01

    Bioluminescent bacterial bioreporters harbor a fusion of bacterial bioluminescence genes (luxCDABE), acting as the reporting element, to a stress-response promoter, serving as the sensing element. Upon exposure to conditions that activate the promoter, such as an environmental stress or the presence of an inducing chemical, the promoter::reporter fusion generates a dose-dependent bioluminescent signal. In order to improve bioluminescent bioreporter performance we have split the luxCDABE genes of Photorhabdus luminescens into two smaller functional units: luxAB, that encode for the luciferase enzyme, which catalyzes the luminescence reaction, and luxCDE that encode for the enzymatic complex responsible for synthesis of the reaction's substrate, a long-chain aldehyde. The expression of each subunit was put under the control of either an inducible stress-responsive promoter or a synthetic constitutive promoter, and different combinations of the two units were tested for their response to selected chemicals in Escherichia coli. In all cases tested, the split combinations proved to be superior to the native luxCDABE configuration, suggesting an improved efficiency in the transcription and/or translation of two small gene units instead of a larger one with the same genes. The best combination was that of an inducible luxAB and a constitutive luxCDE, indicating that aldehyde availability is limited when the five genes are expressed together in E. coli, and demonstrating that improved biosensor performance may be achieved by rearrangement of the lux operon genes.

  17. The mechanism of electronic excitation in the bacterial bioluminescent reaction

    NASA Astrophysics Data System (ADS)

    Nemtseva, E. V.; Kudryasheva, N. S.

    2007-01-01

    The current state of the problem of formation of the electron-excited product in the chemiluminescent reaction that underlies the bacterial luminescence is analysed. Various schemes of chemical transformations capable of producing a bacterial bioluminescence emitter are presented. The problem of excitation of secondary emitters is considered; two possible mechanisms of their excitation are analysed.

  18. Microtiter plate tests for segregation of bioluminescent bacteria.

    PubMed

    Šimkus, Remigijus; Meškienė, Rita; Ledas, Žilvinas; Baronas, Romas; Meškys, Rolandas

    2016-02-01

    It has been recently shown that bioluminescence imaging can be usefully applied to provide new insights into bacterial self-organization. In this work we employ bioluminescence imaging to record images of nutrient rich liquid cultures of the lux-gene reporter Escherichia coli in microtiter plate wells. The images show that patterns of inhomogenous bioluminescence form along the three-phase contact lines. The paper analyzes the dependencies of the average number of luminous aggregates (clouds) on various environmental factors. In particular, our results show that optimal (neutral) pH and high aeration rates determine the highest mean number of clouds, and that spatiotemporal patterns do not form in the pH buffered suspensions. In addition, a sigmoidal (switch-like) dependence of the number of aggregates on the rate of aeration was observed. The obtained bioluminescence imaging data was interpreted by employing the Keller-Segel-Fisher (KSF) model of chemotaxis and logistic growth, adapted to systems of metabolically flexible (two-state) bacteria. The modified KSF model successfully simulated the observed switch-like responses. The results of the microtiter plate tests and their simulations indicate that the segregation of bacteria with different activities proceeds in the three-phase contact line region.

  19. Bioluminescent system for dynamic imaging of cell and animal behavior

    SciTech Connect

    Hara-Miyauchi, Chikako; Tsuji, Osahiko; Hanyu, Aki; Okada, Seiji; Yasuda, Akimasa; Fukano, Takashi; Akazawa, Chihiro; Nakamura, Masaya; Imamura, Takeshi; Matsuzaki, Yumi; Okano, Hirotaka James; and others

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer We combined a yellow variant of GFP and firefly luciferase to make ffLuc-cp156. Black-Right-Pointing-Pointer ffLuc-cp156 showed improved photon yield in cultured cells and transgenic mice. Black-Right-Pointing-Pointer ffLuc-cp156 enabled video-rate bioluminescence imaging of freely-moving animals. Black-Right-Pointing-Pointer ffLuc-cp156 mice enabled tracking real-time drug delivery in conscious animals. -- Abstract: The current utility of bioluminescence imaging is constrained by a low photon yield that limits temporal sensitivity. Here, we describe an imaging method that uses a chemiluminescent/fluorescent protein, ffLuc-cp156, which consists of a yellow variant of Aequorea GFP and firefly luciferase. We report an improvement in photon yield by over three orders of magnitude over current bioluminescent systems. We imaged cellular movement at high resolution including neuronal growth cones and microglial cell protrusions. Transgenic ffLuc-cp156 mice enabled video-rate bioluminescence imaging of freely moving animals, which may provide a reliable assay for drug distribution in behaving animals for pre-clinical studies.

  20. Monitoring Bloom Dynamics of a Common Coastal Bioluminescent Ctenophore

    DTIC Science & Technology

    2009-09-30

    potential in the coastal zone environment. OBJECTIVES Blooms of bioluminescent jellyfish , especially of Mnemiopsis leidyi, are a common occurrence... jellyfish populations are done with net collections by hand at stations weekly, monthly, or seasonally. These time scales severely limit our knowledge...the collection of both biotic and abiotic data continuously. 5 IMPACT/APPLICATIONS As incidents of jellyfish blooms, especially Mnemiopsis

  1. Semi-automated Image Processing for Preclinical Bioluminescent Imaging

    PubMed Central

    Slavine, Nikolai V; McColl, Roderick W

    2015-01-01

    Objective Bioluminescent imaging is a valuable noninvasive technique for investigating tumor dynamics and specific biological molecular events in living animals to better understand the effects of human disease in animal models. The purpose of this study was to develop and test a strategy behind automated methods for bioluminescence image processing from the data acquisition to obtaining 3D images. Methods In order to optimize this procedure a semi-automated image processing approach with multi-modality image handling environment was developed. To identify a bioluminescent source location and strength we used the light flux detected on the surface of the imaged object by CCD cameras. For phantom calibration tests and object surface reconstruction we used MLEM algorithm. For internal bioluminescent sources we used the diffusion approximation with balancing the internal and external intensities on the boundary of the media and then determined an initial order approximation for the photon fluence we subsequently applied a novel iterative deconvolution method to obtain the final reconstruction result. Results We find that the reconstruction techniques successfully used the depth-dependent light transport approach and semi-automated image processing to provide a realistic 3D model of the lung tumor. Our image processing software can optimize and decrease the time of the volumetric imaging and quantitative assessment. Conclusion The data obtained from light phantom and lung mouse tumor images demonstrate the utility of the image reconstruction algorithms and semi-automated approach for bioluminescent image processing procedure. We suggest that the developed image processing approach can be applied to preclinical imaging studies: characteristics of tumor growth, identify metastases, and potentially determine the effectiveness of cancer treatment. PMID:26618187

  2. Novel rat tail discitis model using bioluminescent Staphylococcus aureus.

    PubMed

    Bostian, Phillip A; Karnes, Jonathan M; Cui, Shari; Robinson, Lisa J; Daffner, Scott D; Witt, Michelle R; Emery, Sanford E

    2016-12-05

    Management of spondylodiscitis is a challenging clinical problem requiring medical and surgical treatment strategies. The purpose of this study was to establish a rat model of spondylodiscitis that utilizes bioluminescent Staphylococcus aureus (S. aureus), thus permitting in vivo surveillance of infection intensity. Inocula of the bioluminescent S. aureus strain XEN36 were created in concentrations of 10(2) CFU/0.1 ml, 10(4)  CFU/0.1 ml, and 10(6)  CFU/0.1 ml. Three groups of rats were injected with the bacteria in the most proximal intervertebral tail segment. The third most proximal tail segment was injected with saline as a control. Bioluminescence was measured at baseline, 3 days, and weekly for a total of 6 weeks. Detected bioluminescence for each group peaked at day 3 and returned to baseline in 21 days. The average intensity was highest for the experimental group injected with the most concentrated bacterial solution (10(6)  CFU/0.1 ml). Radiographic analysis revealed loss of intervertebral disc space and evidence of osseous bridging. Saline-injected spaces exhibited no decrease in intervertebral spacing as compared to distal sites. Histologic analysis revealed neutrophilic infiltrates, destruction of the annulus fibrosus and nucleus pulposus, destruction of vertebral endplates, and osseous bridging. Saline-injected discs exhibited preserved annulus fibrosus and nucleus pulposus on histology. This study demonstrates that injection of bioluminescent S. aureus into the intervertebral disc of a rat tail is a viable animal model for spondylodiscitis research. This model allows for real-time, in vivo quantification of infection intensity, which may decrease the number of animals required for infection studies of the intervertebral disc. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

  3. 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.

  4. Validation of constitutively expressed bioluminescent Pseudomonas aeruginosa as a rapid microbiological quantification tool.

    PubMed

    Shah, N; Naseby, D C

    2015-06-15

    Whole cell biosensors have been extensively used for monitoring toxicity and contamination of various compounds and xenobiotics in environmental biology and microbial ecology; their application in the pharmaceutical and cosmetics industries has been limited. According to several pharmacopoeias, pharmaceutical products must be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. However there is a lack of a validated bioluminescence method. Prototype whole cell microbial biosensors have already been developed in Pseudomonas aeruginosa ATCC 9027. Validation of the bioluminescent strains was performed in accordance with the pharmacopoeia, Parenteral Drug Association and International Organisation of Standardisation. These strains demonstrated that the bioluminescent method was accurate, precise and equivalent, as compared with plate counting at a range of 10(3)-10(7) CFU/mL. Percentage recoveries using the bioluminescent method were between 70% and 130% for all bioluminescent strains and therefore the bioluminescent method was accurate according to the criteria set in PDA technical report 33. The method was also more precise (relative standard deviation less than 15%) than the traditional plate counting method or the ATP bioluminescent method. The lower limit of detection was 10(3) CFU/mL. Two-way ANOVA showed no significant difference between the traditional plate counting and the novel bioluminescent method for all bioluminescent strains. The bioluminescent constructs passed/exceeded pharmacopoeia-specified criteria for range, limit of detection, accuracy, precision and equivalence.

  5. Enhanced operation of femtosecond lasers and applications in cell transfection.

    PubMed

    Brown, Christian T A; Stevenson, David J; Tsampoula, Xanthi; McDougall, Craig; Lagatsky, Alexander A; Sibbett, Wilson; Gunn-Moore, Frank J; Dholakia, Kishan

    2008-08-01

    In this work we present a review and discussion on the enhancement of femtosecond (fs) lasers for use within biophotonics with a particular focus on their use in optical transfection techniques. We describe the broad range of source options now available for the generation of femtosecond pulses before briefly reviewing the application of fs laser in optical transfection studies. We show that major performance enhancements may be obtained by optimising the spatial and temporal performance of the laser source before considering possible future directions in this field. In relation to optical transfection we describe how such laser sources initiate a multiphoton process to permeate the cell membrane in a transient fashion. We look at aspects of this technique including the ability to combine transfection with optical trapping. For future implementation of such transfection we explore the role of new sources and "nondiffracting" light fields.

  6. Influence of antibiotic pressure on bacterial bioluminescence, with emphasis on Staphylococcus aureus.

    PubMed

    Daghighi, Seyedmojtaba; Sjollema, Jelmer; Harapanahalli, Akshay; Dijkstra, Rene J B; van der Mei, Henny C; Busscher, Henk J

    2015-12-01

    Bioluminescence imaging is used for longitudinal evaluation of bacteria in live animals. Clear relations exist between bacterial numbers and their bioluminescence. However, bioluminescence images of Staphylococcus aureus Xen29, S. aureus Xen36 and Escherichia coli Xen14 grown on tryptone soy agar in Etests demonstrated increased bioluminescence at sub-MICs of different antibiotics. This study aimed to further evaluate the influence of antibiotic pressure on bioluminescence in S. aureus Xen29. Bioluminescence of S. aureus Xen29, grown planktonically in tryptone soy broth, was quantified in the absence and presence of different concentrations of vancomycin, ciprofloxacin, erythromycin or chloramphenicol and was related to expression of the luxA gene under antibiotic pressure measured using real-time PCR. In the absence of antibiotics, staphylococcal bioluminescence increased over time until a maximum after ca. 6h of growth, and subsequently decreased to the detection threshold after 24h of growth owing to reduced bacterial metabolic activity. Up to MICs of the antibiotics, bioluminescence increased according to a similar pattern up to 6h of growth, but after 24h bioluminescence was higher than in the absence of antibiotics. Contrary to expectations, bioluminescence per organism (CFU) after different growth periods in the absence and at MICs of different antibiotics decreased with increasing expression of luxA. Summarising, antibiotic pressure impacts the relation between CFU and bioluminescence. Under antibiotic pressure, bioluminescence is not controlled by luxA expression but by co-factors impacting the bacterial metabolic activity. This conclusion is of utmost importance when evaluating antibiotic efficacy in live animals using bioluminescent bacterial strains.

  7. Non-Viral, Lipid-Mediated DNA and mRNA Gene Therapy of the Central Nervous System (CNS): Chemical-Based Transfection.

    PubMed

    Hecker, James G

    2016-01-01

    Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Here, we describe techniques for cationic lipid-mediated delivery of nucleic acids encoding reporter genes in a variety of cell lines. We describe optimized formulations and transfection procedures that we previously assessed by bioluminescence and flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in non-dividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. Duration of expression in eukaryotic cells after mRNA transcript delivery depends on multiple factors, including transcript stability, protein turnover, and cell type. Delivery of DNA results in onset of expression within 5 h after transfection, a peak in expression 24-48 h after transfection, and a return to baseline that can be as long as several weeks after transfection. In vitro results are consistent with our in vivo delivery results, techniques for which are described as well. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression and greater efficiency, particularly in non-dividing cells, while the longer duration and

  8. Modelling dinoflagellates as an approach to the seasonal forecasting of bioluminescence in the North Atlantic

    NASA Astrophysics Data System (ADS)

    Marcinko, Charlotte L. J.; Martin, Adrian P.; Allen, John T.

    2014-11-01

    Bioluminescence within ocean surface waters is of significant interest because it can enhance the study of subsurface movement and organisms. Little is known about how bioluminescence potential (BPOT) varies spatially and temporally in the open ocean. However, light emitted from dinoflagellates often dominates the stimulated bioluminescence field. As a first step towards forecasting surface ocean bioluminescence in the open ocean, a simple ecological model is developed which simulates seasonal changes in dinoflagellate abundance. How forecasting seasonal changes in BPOT may be achieved through combining such a model with relationships derived from observations is discussed and an example is given. The study illustrates a potential new approach to forecasting BPOT through explicitly modelling the population dynamics of a prolific bioluminescent phylum. The model developed here offers a promising platform for the future operational forecasting of the broad temporal changes in bioluminescence within the North Atlantic. Such forecasting of seasonal patterns could provide valuable information for the targeting of scientific field campaigns.

  9. Activity of upper electron-excited states in bioluminescence of coelenterates

    NASA Astrophysics Data System (ADS)

    Belogurova, N. V.; Alieva, R. R.; Kudryasheva, N. S.

    2009-04-01

    The involvement of upper electron-excited states as the primary excited states into bioluminescence of coelenterates was experimentally verified. A series of fluorescent molecules was used as foreign energy acceptors in this bioluminescent reaction. The fluorescent aromatic compounds - pyrene, 2-methoxy-naphtalene, naphthalene, and 1,4-diphenylbutadiene - were selected, with fluorescent state energies ranging from 26,700 to 32,500 cm -1. Excitation of these molecules by Forster singlet-singlet energy transfer from S of bioluminescence emitter and by light absorption were excluded. The weak sensitized fluorescence of three compounds was found in the course of bioluminescent reaction. Energy of the upper electron-excited states of the bioluminescent emitter was located around 31,000 cm -1. Localization of the primary excitation on a carbonyl group of coelenteramide molecule is discussed. Comparison of the primary excitation in bioluminescent processes of coelenterates and bacteria is provided.

  10. Hydrophobic Moiety of Cationic Lipids Strongly Modulates Their Transfection Activity

    SciTech Connect

    Koynova, Rumiana; Tenchov, Boris; Wang, Li; MacDonald, Robert C.

    2010-01-18

    Synthetic cationic lipids are widely used components of nonviral gene carriers, and the factors regulating their transfection efficiency are the subject of considerable interest. In view of the important role that electrostatic interactions with the polyanionic nucleic acids play in formation of lipoplexes, a common empirical approach to improving transfection has been the synthesis and testing of amphiphiles with new versions of positively charged polar groups, while much less attention has been given to the role of the hydrophobic lipid moieties. On the basis of data for {approx}20 cationic phosphatidylcholine (PC) derivatives, here we demonstrate that hydrocarbon chain variations of these lipids modulate by over 2 orders of magnitude their transfection efficiency. The observed molecular structure-activity relationship manifests in well-expressed dependences of activity on two important molecular characteristics, chain unsaturation and total number of carbon atoms in the lipid chains, which is representative of the lipid hydrophobic volume and hydrophilic-lipophilic ratio. Transfection increases with decrease of chain length and increase of chain unsaturation. Maximum transfection was found for cationic PCs with monounsaturated 14:1 chains. It is of particular importance that the high-transfection lipids strongly promote cubic phase formation in zwitterionic membrane phosphatidylethanolamine (PE). These remarkable correlations point to an alternative, chain-dependent process in transfection, not related to the electrostatic cationic-anionic lipid interactions.

  11. [Transfection of HL-60 cells by Venus lentiviral vector].

    PubMed

    Li, Zheng; Hu, Shao-Yan; Cen, Jian-Nong; Chen, Zi-Xing

    2013-06-01

    In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.

  12. Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA.

    PubMed

    Boizet, B; Flickinger, J L; Chassy, B M

    1988-12-01

    A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.

  13. A reverse transfection technology to genetically engineer adult stem cells.

    PubMed

    Okazaki, Arimichi; Jo, Jun-Ichiro; Tabata, Yasuhiko

    2007-02-01

    A new non-viral method of gene transfection was designed to enhance the level of gene expression for rat mesenchymal stem cells (MSCs). Pullulan was cationized using chemical introduction of spermine to prepare cationized pullulan of non-viral carrier (spermine-pullulan). The spermine-pullulan was complexed with a plasmid deoxyribonucleic acid (DNA) of luciferase and coated on the surface of culture substrate together with Pronectin of artificial cell adhesion protein. MSCs were cultured and transfected on the complex-coated substrate (reverse transfection), and the level and duration of gene expression were compared with those of MSCs transfected by culturing in the medium containing the plasmid DNA-spermine-pullulan complex (conventional method). The reverse transfection method enhanced and prolonged gene expression significantly more than did the conventional method. The reverse method permitted the transfection culture of MSCs in the presence of serum, in contrast to the conventional method, which gave cells a good culture condition to lower cytotoxicity. The reverse transfection was carried out for a non-woven fabric of polyethylene terephthalate (PET) coated with the complex and Pronectin using agitation and stirring culture methods. The two methods enhanced the level and duration of gene expression for MSCs significantly more than did the static method. It is possible that medium circulation improves the culture conditions of cells in terms of oxygen and nutrition supply and waste excretion, resulting in enhanced gene expression.

  14. Similarity states of homogeneous stably-stratified turbulence at infinite Froude number

    NASA Technical Reports Server (NTRS)

    Chasnov, Jeffrey R.

    1993-01-01

    We present evidence of similarity states which may develop inhomogeneous stably-stratified flows if a dimensionless group in addition to the Reynolds number, the so-called Froude number, is sufficiently large. Here, we define the Froude number as the ratio of the internal wave time-scale to the turbulence time-scale. We examine three different similarity states which may develop depending on the initial conditions of the velocity and density fields. Theoretical arguments and results of large-eddy simulations will be presented. We will conclude this report with some speculative thoughts on similarity states which may develop in stably-stratified turbulence at arbitrary Froude number as well as our future research plans in this area.

  15. Turbulent circulation above the surface heat source in stably stratified atmosphere

    NASA Astrophysics Data System (ADS)

    Kurbatskii, A. F.; Kurbatskaya, L. I.

    2016-10-01

    The 3-level RANS approach for simulating a turbulent circulation over the heat island in a stably stratified environment under nearly calm conditions is formulated. The turbulent kinetic energy its spectral consumption (dissipation) and the dispersion of turbulent fluctuations of temperature are found from differential equations, thus the correct modeling of transport processes in the interface layer with the counter-gradient heat flux is assured. The three-parameter turbulence RANS approach minimizes difficulties in simulating the turbulent transport in a stably stratified environment and reduces efforts needed for the numerical implementation of the 3-level RANS approach. Numerical simulation of the turbulent structure of the penetrative convection over the heat island under conditions of stably stratified atmosphere demonstrates that the three-equation model is able to predict the thermal circulation induced by the heat island. The temperature distribution, root-mean-square fluctuations of the turbulent velocity and temperature fields and spectral turbulent kinetic energy flux are in good agreement with the experimental data. The model describes such thin physical effects, as a crossing of vertical profiles of temperature of a thermal plume with the formation of the negative buoyancy area testifying to development of the dome-shaped form at the top part of a plume in the form of "hat".

  16. Identifying stably expressed genes from multiple RNA-Seq data sets

    PubMed Central

    Emerson, Sarah; Chang, Jeff H.; Di, Yanming

    2016-01-01

    We examined RNA-Seq data on 211 biological samples from 24 different Arabidopsis experiments carried out by different labs. We grouped the samples according to tissue types, and in each of the groups, we identified genes that are stably expressed across biological samples, treatment conditions, and experiments. We fit a Poisson log-linear mixed-effect model to the read counts for each gene and decomposed the total variance into between-sample, between-treatment and between-experiment variance components. Identifying stably expressed genes is useful for count normalization and differential expression analysis. The variance component analysis that we explore here is a first step towards understanding the sources and nature of the RNA-Seq count variation. When using a numerical measure to identify stably expressed genes, the outcome depends on multiple factors: the background sample set and the reference gene set used for count normalization, the technology used for measuring gene expression, and the specific numerical stability measure used. Since differential expression (DE) is measured by relative frequencies, we argue that DE is a relative concept. We advocate using an explicit reference gene set for count normalization to improve interpretability of DE results, and recommend using a common reference gene set when analyzing multiple RNA-Seq experiments to avoid potential inconsistent conclusions. PMID:28028467

  17. [VEGF gene expression in transfected human multipotent stromal cells].

    PubMed

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-01-01

    Dynamics of VEGF gene expression in transfected multipotent stromal cells from adipose tissue was examined using electroporation and lipofection. Differences in the potency and dynamics of plasmid elimination (up to day 9) between cell cultures were observed. All cultures were divided into fast and slow plasmid-eliminating ones. Interculture differences in VEGF expression were detected. The possibility of a 5-6-fold increase of VEGF expression was shown. There were no differences in transfection potency, plasmid elimination dynamics, and VEGF expression after transfection by both nonviral methods.

  18. Lipid-based transfection reagents can interfere with cholesterol biosynthesis.

    PubMed

    Danielli, Mauro; Marinelli, Raúl A

    2016-02-15

    Lipid-based transfection reagents are widely used for delivery of small interfering RNA into cells. We examined whether the commonly used commercial transfection reagents DharmaFECT-4 and Lipofectamine 2000 can interfere with lipid metabolism by studying cholesterogenesis. Cholesterol de novo synthesis from [(14)C]acetate was assessed in human hepatocyte-derived Huh-7 cells. The results revealed that DharmaFECT, but not Lipofectamine, markedly inhibited cholesterol biosynthesis by approximately 70%. Cell viability was not significantly altered. These findings suggest that caution is required in the choice of certain lipid-based transfection reagents for gene silencing experiments, particularly when assessing cholesterol metabolism.

  19. Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

    PubMed Central

    Hansen, Louise L.; van Ooijen, Gerben

    2016-01-01

    The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself. Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background. PMID:27684315

  20. Phage-amplified bioluminescent bioreporters for the detection of foodborne pathogens

    NASA Astrophysics Data System (ADS)

    Ripp, Steven; Young, Jacque C.; Ozen, Aysu; Jegier, Patricia; Johnson, Courtney; Daumer, Kathleen; Garland, Jay; Sayler, Gary S.

    2004-06-01

    The objective of this investigation is to develop a bioluminescent bioreporter system for the detection and monitoring of pathogenic microbial species. Current detection methodologies typically rely on time-consuming sample pre-enrichment steps to elevate pathogen concentrations to detectable levels or DNA based polymerase chain reaction (PCR) techniques that require extensive user training and expensive instrumentation. Detection utilizing bioluminescent bioreporter organisms, however, can provide a simple and rapid means of monitoring foodborne pathogens. Bioluminescent bioreporters are engineered to produce light in response to specific environmental inducers. The light signal is then measured with photodetector devices to generate a quantitative assessment of inducer concentration. The immediate goal of this research effort is to integrate key quorum sensing signal transduction elements into pathogen specific bacteriophages. Upon infection of a unique pathogenic species by the bacteriophages, quorum sensing signals will be generated that will subsequently stimulate bioluminescence in neighboring bioluminescent bioreporter cells. Utilizing both bacteriophages and bioluminescent bioreporters, we realize exceptional pathogen specificity while attaining enhanced bioluminescence production. This integrative approach will lead to rapid pathogen identification without requisite sample pre-enrichment. Additionally, since the bioluminescent response is completely intrinsic to the bioreporter organism, no user interventions are required for generating light signals; the protocol requires only addition of the food sample with the bacteriophage/bioluminescent bioreporter system. Measurement of light responses can be achieved using high-throughput microtiter plate readers, hand-held photomultiplier units, or microchip luminometers.

  1. Measurement of Bacterial Bioluminescence Intensity and Spectrum: Current Physical Techniques and Principles.

    PubMed

    Jia, Kun; Ionescu, Rodica Elena

    2016-01-01

    : Bioluminescence is light production by living organisms, which can be observed in numerous marine creatures and some terrestrial invertebrates. More specifically, bacterial bioluminescence is the "cold light" produced and emitted by bacterial cells, including both wild-type luminescent and genetically engineered bacteria. Because of the lively interplay of synthetic biology, microbiology, toxicology, and biophysics, different configurations of whole-cell biosensors based on bacterial bioluminescence have been designed and are widely used in different fields, such as ecotoxicology, food toxicity, and environmental pollution. This chapter first discusses the background of the bioluminescence phenomenon in terms of optical spectrum. Platforms for bacterial bioluminescence detection using various techniques are then introduced, such as a photomultiplier tube, charge-coupled device (CCD) camera, micro-electro-mechanical systems (MEMS), and complementary metal-oxide-semiconductor (CMOS) based integrated circuit. Furthermore, some typical biochemical methods to optimize the analytical performances of bacterial bioluminescent biosensors/assays are reviewed, followed by a presentation of author's recent work concerning the improved sensitivity of a bioluminescent assay for pesticides. Finally, bacterial bioluminescence as implemented in eukaryotic cells, bioluminescent imaging, and cancer cell therapies is discussed.

  2. Bioluminescence in the Ocean: Origins of Biological, Chemical, and Ecological Diversity

    NASA Astrophysics Data System (ADS)

    Widder, E. A.

    2010-05-01

    From bacteria to fish, a remarkable variety of marine life depends on bioluminescence (the chemical generation of light) for finding food, attracting mates, and evading predators. Disparate biochemical systems and diverse phylogenetic distribution patterns of light-emitting organisms highlight the ecological benefits of bioluminescence, with biochemical and genetic analyses providing new insights into the mechanisms of its evolution. The origins and functions of some bioluminescent systems, however, remain obscure. Here, I review recent advances in understanding bioluminescence in the ocean and highlight future research efforts that will unite molecular details with ecological and evolutionary relationships.

  3. Monitoring of Bioluminescent Lactobacillus plantarum in a Complex Food Matrix

    PubMed Central

    Narbad, Arjan

    2017-01-01

    A bioluminescent Lactobacillus plantarum (pLuc2) strain was constructed. The luminescent signal started to increase during the early exponential phase and reached its maximum in the mid-exponential phase in a batch culture of the strain. The signal detection sensitivity of the strain was the highest in PBS (phosphate buffered saline), followed by milk and MRS broth, indicating that the sensitivity was influenced by the matrix effect. The strain was used in millet seed fermentation which has a complex matrix and native lactic acid bacteria (LAB). The luminescent signal was gradually increased until 9 h during fermentation and abolished at 24 h, indicating that the strain could be specifically tracked in the complex matrix and microflora. Therefore, the bioluminescent labeling system can be used for monitoring LAB in food and dairy sciences and industries. PMID:28316482

  4. Fluorescence and Bioluminescence Imaging of Orthotopic Brain Tumors in Mice.

    PubMed

    McKinnon, Emilie; Moore, Alfred; Dixit, Suraj; Zhu, Yun; Broome, Ann-Marie

    2017-01-01

    Optical imaging strategies, such as fluorescence and bioluminescence imaging, are non-invasive, in vivo whole body imaging techniques utilized to study cancer. Optical imaging is widely used in preclinical work because of its ease of use and cost-friendliness. It also provides the opportunity to study animals and biological responses longitudinally over time. Important considerations include depth of tissue penetration, photon scattering, absorption and the choice of light emitting probe, all of which affect the resolution (image quality and data information) and the signal to noise ratio of the image. We describe how to use bioluminescence and fluorescence imaging to track a chemotherapeutic delivery nanocarrier conjugated with a fluorophore to determine its localization in vivo.

  5. Monitoring of Bioluminescent Lactobacillus plantarum in a Complex Food Matrix.

    PubMed

    Moon, Gi-Seong; Narbad, Arjan

    2017-01-01

    A bioluminescent Lactobacillus plantarum (pLuc2) strain was constructed. The luminescent signal started to increase during the early exponential phase and reached its maximum in the mid-exponential phase in a batch culture of the strain. The signal detection sensitivity of the strain was the highest in PBS (phosphate buffered saline), followed by milk and MRS broth, indicating that the sensitivity was influenced by the matrix effect. The strain was used in millet seed fermentation which has a complex matrix and native lactic acid bacteria (LAB). The luminescent signal was gradually increased until 9 h during fermentation and abolished at 24 h, indicating that the strain could be specifically tracked in the complex matrix and microflora. Therefore, the bioluminescent labeling system can be used for monitoring LAB in food and dairy sciences and industries.

  6. Rapid, sensitive bioluminescent reporter technology for napthalene exposure and biodegradation

    SciTech Connect

    King, J.M.H.; DiGrazia, P.M.; Applegate, B.; Burlage, R.; Sanseverino, J.; Dunbar, P.; Sayler, G.S. ); Larimer, F. )

    1990-08-17

    A bioluminescent reporter plasmid for naphthalene catabolism (pUTK21) was developed by transposon (Tn4431) insertion of the lux gene cassette from Vibrio fischeri into a naphthalene catabolic plasmid in Pseudomonas fluorescens. The insertion site of the lux transposon was the nahG gene encoding for salicylate hydroxylase. Luciferase-mediated light production from P. fluorescens strains harboring this plasmid was induced on exposure to naphthalene or the regulatory inducer metabolite, salicylate. In continuous culture, light induction was rapid and was highly responsive to dynamic changes in naphthalene exposure. Strains harboring pUTK21 were responsive to aromatic hydrocarbon contamination in Manufactured Gas Plant soils and produced sufficient light to serve as biosensors of naphthalene exposure and reporters of napthalene biodegradative activity. The robust and sensitive nature of the bioluminescent reporter technology suggests that new sensing methods can be developed for on-line process monitoring and control in complex environmental matrices.

  7. Structural basis for the spectral difference in luciferase bioluminescence.

    PubMed

    Nakatsu, Toru; Ichiyama, Susumu; Hiratake, Jun; Saldanha, Adrian; Kobashi, Nobuyuki; Sakata, Kanzo; Kato, Hiroaki

    2006-03-16

    Fireflies communicate with each other by emitting yellow-green to yellow-orange brilliant light. The bioluminescence reaction, which uses luciferin, Mg-ATP and molecular oxygen to yield an electronically excited oxyluciferin species, is carried out by the enzyme luciferase. Visible light is emitted during relaxation of excited oxyluciferin to its ground state. The high quantum yield of the luciferin/luciferase reaction and the change in bioluminescence colour caused by subtle structural differences in luciferase have attracted much research interest. In fact, a single amino acid substitution in luciferase changes the emission colour from yellow-green to red. Although the crystal structure of luciferase from the North American firefly (Photinus pyralis) has been described, the detailed mechanism for the bioluminescence colour change is still unclear. Here we report the crystal structures of wild-type and red mutant (S286N) luciferases from the Japanese Genji-botaru (Luciola cruciata) in complex with a high-energy intermediate analogue, 5'-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine (DLSA). Comparing these structures to those of the wild-type luciferase complexed with AMP plus oxyluciferin (products) reveals a significant conformational change in the wild-type enzyme but not in the red mutant. This conformational change involves movement of the hydrophobic side chain of Ile 288 towards the benzothiazole ring of DLSA. Our results indicate that the degree of molecular rigidity of the excited state of oxyluciferin, which is controlled by a transient movement of Ile 288, determines the colour of bioluminescence during the emission reaction.

  8. Dynamic Modeling of Marine Bioluminescence and Night Time Leaving Radiance

    DTIC Science & Technology

    2010-09-30

    2003 by using BP data from four AUVs sections: DORADO sections taken 13 August and 14 August, and AUV REMUS sections taken on 14 August (Figure 1...and AUV REMUS data. 3 Figure 1. (A) V-shaped transect of CalPoly AUV REMUS and sections sampled by AUV DORADO ; (B) AUV REMUS observed...chlorophyll, backscattering and bioluminescence during 11-15 August. Solid vertical lines indicate location of the M1 mooring; (C) AUV DORADO observed

  9. Bioluminescent bacteria as indicators of chemical contamination of coastal waters.

    PubMed

    Frischer, M E; Danforth, J M; Foy, T F; Juraske, R

    2005-01-01

    The ratio of bioluminescent to total bacteria (bioluminescent ratio, BLR) as an indicator of a variety of types of anthropogenic contamination of estuarine ecosystems was evaluated through a series of laboratory and field studies. Laboratory studies indicated that the BLR of natural bacterioplankton communities was proportionally reduced in the presence of a number of contaminants including diesel fuel and saltmarsh sediments co-contaminated with mercury and polychlorinated biphenyls (PCBs). Bioluminescent ratio inhibition was observed after short-term exposure to a contaminant suggesting a physiological rather than a population response of native microbial communities. Simulated eutrophication did not suppress the BLR. Field observations of the BLR were conducted weekly for a 2-yr period in the Skidaway River estuary, Georgia, USA. These observations revealed considerable seasonal variability associated with the BLR. Bioluminescent ratios were highest during the summer (25 +/- 15%), lower in the fall (6 +/- 5%) and spring (3 +/- 2%), and near zero during the winter. Although the BLR was not significantly correlated to salinity at a single site (Skidaway River estuary), the BLR was significantly correlated with salinity when sites within the same estuary system were compared (r2 = 0.93). Variation in BLR was not correlated to standard bacteriological indicators of water quality including total and fecal coliform bacteria. Comparison of the BLR from impacted and pristine estuarine sites during the fall suggested that anthropogenically impacted sites exhibited lower BLR than predicted from salinity versus BLR relationships developed in pristine systems. These observations suggest that the BLR could be used as a simple and reliable initial indicator of chemical contamination of estuarine systems resulting from human activity.

  10. Bacterial bioluminescence and Gumbel statistics: From quorum sensing to correlation

    NASA Astrophysics Data System (ADS)

    Delle Side, Domenico; Velardi, Luciano; Nassisi, Vincenzo; Pennetta, Cecilia; Alifano, Pietro; Talà, Adelfia; Salvatore Tredici, Maurizio

    2013-12-01

    We show that, in particular experimental conditions, the time course of the radiant fluxes, measured from a bioluminescent emission of a Vibrio harveyi related strain, collapse after suitable rescaling onto the Gumbel distribution of extreme value theory. We argue that the activation times of the strain luminous emission follow the universal behavior described by this statistical law, in spite of the fact that no extremal process is known to occur.

  11. Reverse transfected cell microarrays in infectious disease research.

    PubMed

    Konrad, Andreas; Jochmann, Ramona; Kuhn, Elisabeth; Naschberger, Elisabeth; Chudasama, Priya; Stürzl, Michael

    2011-01-01

    Several human pathogenic viruses encode large genomes with often more than 100 genes. Viral pathogenicity is determined by carefully orchestrated co-operative activities of several different viral genes which trigger the phenotypic functions of the infected cells. Systematic analyses of these complex interactions require high-throughput transfection technology. Here we have provided a laboratory manual for the reverse transfected cell microarray (RTCM; alternative name: cell chip) as a high-throughput transfection procedure, which has been successfully applied for the systematic analyses of single and combination effects of genes encoded by the human herpesvirus-8 on the NF-kappaB signal transduction pathway. In order to quantitatively determine the effects of viral genes in transfected cells, protocols for the use of GFP as an indicator gene and for indirect immunofluorescence staining of cellular target proteins have been included. RTCM provides a useful methodological approach to investigate systematically combination effects of viral genes on cellular functions.

  12. Detection of Organic Compounds with Whole-Cell Bioluminescent Bioassays

    PubMed Central

    Xu, Tingting; Close, Dan; Smartt, Abby; Ripp, Steven

    2015-01-01

    Natural and manmade organic chemicals are widely deposited across a diverse range of ecosystems including air, surface water, groundwater, wastewater, soil, sediment, and marine environments. Some organic compounds, despite their industrial values, are toxic to living organisms and pose significant health risks to humans and wildlife. Detection and monitoring of these organic pollutants in environmental matrices therefore is of great interest and need for remediation and health risk assessment. Although these detections have traditionally been performed using analytical chemical approaches that offer highly sensitive and specific identification of target compounds, these methods require specialized equipment and trained operators, and fail to describe potential bioavailable effects on living organisms. Alternatively, the integration of bioluminescent systems into whole-cell bioreporters presents a new capacity for organic compound detection. These bioreporters are constructed by incorporating reporter genes into catabolic or signaling pathways that are present within living cells and emit a bioluminescent signal that can be detected upon exposure to target chemicals. Although relatively less specific compared to analytical methods, bioluminescent bioassays are more cost-effective, more rapid, can be scaled to higher throughput, and can be designed to report not only the presence but also the bioavailability of target substances. This chapter reviews available bacterial and eukaryotic whole-cell bioreporters for sensing organic pollutants and their applications in a variety of sample matrices. PMID:25084996

  13. Relationship between stability and bioluminescence color of firefly luciferase.

    PubMed

    Maghami, Parvaneh; Ranjbar, Bijan; Hosseinkhani, Saman; Ghasemi, Atiyeh; Moradi, Ali; Gill, Pooria

    2010-03-01

    Firefly luciferase catalyzes the oxidation of luciferin in the presence of ATP, Mg(2+) and molecular oxygen. The bioluminescence color of firefly luciferases is identified by the luciferase structure and assay conditions. Amongst different types of beetles, luciferase from Phrixotrix railroad worm (PhRE) with a unique additional residue (Arg353) naturally emits red bioluminescence color. By insertion of Arg356 in luciferase of Lampyris turkestanicus, corresponding to Arg353 in Phrixotrix hirtus, the color of the emitted light was changed to red. To understand the effect of this position on the bioluminescence color shift, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gln) were inserted into Photinus pyralis luciferase. Comparison of mutants with native luciferase shows that mutation brought an increase in the content of secondary structure and globular compactness of (P. pylalis) luciferase. Comparative study of chemical denaturation of native and mutant luciferases by activity measurement, intrinsic and extrinsic fluorescence, circular dichroism, and DSC techniques revealed that insertion of positively charged residues (Arg, Lys) in the flexible loop (352-358) plays a significant role on the stability of (P. pyralis) luciferase and changes the light color to red.

  14. Image-guided simulation for bioluminescence tomographic imaging

    NASA Astrophysics Data System (ADS)

    Kumar, Durairaj; Cong, Wenxiang; Thiesse, Jacqueline; Nixon, Earl; Meinel, John, Jr.; Cong, Alex; McLennan, Geoffrey; Hoffman, Eric A.; Ming, Jiang; Wang, Ge

    2005-04-01

    Noninvasive imaging of the reporter gene expression based on bioluminescence is playing an important role in the areas of cancer biology, cell biology, and gene therapy. The central problem for the bioluminescence tomography (BLT) we are developing is to reconstruct the underlying bioluminescent source distribution in a small animal using a modality fusion approach. To solve this inversion problem, a mathematical model of the mouse is built from a CT/micro-CT scan, which enables the assignment of optical parameters to various regions in the model. This optical geometrical model is used in the Monte Carlo simulation to calculate the flux distribution on the animal body surface, as a key part of the BLT process. The model development necessitates approximations in surface simplification, and so on. It leads to the model mismatches of different kinds. To overcome such discrepancies, instead of developing a mathematical model, segmented CT images are directly used in our simulation software. While the simulation code is executed, those images that are relevant are assessed according to the location of the propagating photon. Depending upon the segmentation rules including the pixel value range, appropriate optical parameters are selected for statistical sampling of the free path and weight of the photon. In this paper, we report luminescence experiments using a physical mouse phantom to evaluate this image-guided simulation procedure, which suggest both the feasibility and some advantages of this technique over the existing methods.

  15. Catabolic gene expression is monitored by bioluminescence in bioreactor studies

    SciTech Connect

    Burlage, R.S.; Kuo, D.; Palumbo, A.V.

    1993-01-01

    In order to study the expression of specific catabolic genes under defined conditions, and to determine whether certain conditions tend to increase or decrease metal catabolic activities, a bioreporter gene can be introduced into the microorganism. Activity from such bioreporter gene would indicate successful bioremediation. Our laboratory has produced several bioreporter strains using the bioluminescent lux genes of Vibrio fischeri. A bioreporter producing visible light when genetic expression is induced. The bioluminescent system include sensitivity of detection, analysis of response in real- time, and on-line capability. We constructed a bioreporter strain aimed at following the degradation of toluene and related compounds in order to study expression of the catabolic genes with various substrates and under optimized bioreactor conditions. We have been able to detect the induction of a specific operon in response to the addition of oxylene, as a gratuitous inducer of the catabolic genes. A strong bioluminescent signal in these studies. We have varied the medium of an induced bioreactor culture of RB1401, and our data suggest that conditions for optimal expression of the catabolic operon might not be identical with optimal growth conditions.

  16. Catabolic gene expression is monitored by bioluminescence in bioreactor studies

    SciTech Connect

    Burlage, R.S.; Kuo, D.; Palumbo, A.V.

    1993-03-01

    In order to study the expression of specific catabolic genes under defined conditions, and to determine whether certain conditions tend to increase or decrease metal catabolic activities, a bioreporter gene can be introduced into the microorganism. Activity from such bioreporter gene would indicate successful bioremediation. Our laboratory has produced several bioreporter strains using the bioluminescent lux genes of Vibrio fischeri. A bioreporter producing visible light when genetic expression is induced. The bioluminescent system include sensitivity of detection, analysis of response in real- time, and on-line capability. We constructed a bioreporter strain aimed at following the degradation of toluene and related compounds in order to study expression of the catabolic genes with various substrates and under optimized bioreactor conditions. We have been able to detect the induction of a specific operon in response to the addition of oxylene, as a gratuitous inducer of the catabolic genes. A strong bioluminescent signal in these studies. We have varied the medium of an induced bioreactor culture of RB1401, and our data suggest that conditions for optimal expression of the catabolic operon might not be identical with optimal growth conditions.

  17. Robust image modeling technique with a bioluminescence image segmentation application

    NASA Astrophysics Data System (ADS)

    Zhong, Jianghong; Wang, Ruiping; Tian, Jie

    2009-02-01

    A robust pattern classifier algorithm for the variable symmetric plane model, where the driving noise is a mixture of a Gaussian and an outlier process, is developed. The veracity and high-speed performance of the pattern recognition algorithm is proved. Bioluminescence tomography (BLT) has recently gained wide acceptance in the field of in vivo small animal molecular imaging. So that it is very important for BLT to how to acquire the highprecision region of interest in a bioluminescence image (BLI) in order to decrease loss of the customers because of inaccuracy in quantitative analysis. An algorithm in the mode is developed to improve operation speed, which estimates parameters and original image intensity simultaneously from the noise corrupted image derived from the BLT optical hardware system. The focus pixel value is obtained from the symmetric plane according to a more realistic assumption for the noise sequence in the restored image. The size of neighborhood is adaptive and small. What's more, the classifier function is base on the statistic features. If the qualifications for the classifier are satisfied, the focus pixel intensity is setup as the largest value in the neighborhood.Otherwise, it will be zeros.Finally,pseudo-color is added up to the result of the bioluminescence segmented image. The whole process has been implemented in our 2D BLT optical system platform and the model is proved.

  18. Flexible peritoneal windows for quantitative fluorescence and bioluminescence preclinical imaging.

    PubMed

    Souris, Jeffrey S; Hickson, Jonathan A; Msezane, Lambda; Rinker-Schaeffer, Carrie W; Chen, Chin-Tu

    2013-01-01

    At present, there is considerable interest in the use of in vivo fluorescence and bioluminescence imaging to track the onset and progression of pathologic processes in preclinical models of human disease. Optical quantitation of such phenomena, however, is often problematic, frequently complicated by the overlying tissue's scattering and absorption of light, as well as the presence of endogenous cutaneous and subcutaneous fluorophores. To partially circumvent this information loss, we report here the development of flexible, surgically implanted, transparent windows that enhance quantitative in vivo fluorescence and bioluminescence imaging of optical reporters. These windows are metal and glass free and thus compatible with computed tomography, magnetic resonance imaging, positron emission tomography, and single-photon emission computed tomography; they also permit visualization of much larger areas with fewer impediments to animal locomotion and grooming than those previously described. To evaluate their utility in preclinical imaging, we surgically implanted these windows in the abdominal walls of female athymic nude mice and subsequently inoculated each animal with 1 × 10(4) to 1 × 10(6) bioluminescent human ovarian cancer cells (SKOV3ip.1-luc). Longitudinal imaging studies of fenestrated animals revealed up to 48-fold gains in imaging sensitivity relative to nonfenestrated animals, with relatively few complications, allowing wide-field in vivo visualization of nascent metastatic ovarian cancer colonization.

  19. Invariant chain+ N2a neuroblastoma cells stably expressing the class II MHC transactivator CIITA fail to stimulate anti-tumor immunity.

    PubMed

    Rickard, Steve; Ono, Santa Jeremy

    2008-12-01

    A promising cancer treatment strategy involves stimulation of anti-tumor immune responses. CD4(+) T cell responses are particularly desirable, as they enhance CD8(+) T cell activity and provide immune memory. The major histocompatibility complex (MHC) class II transactivator CIITA can be used to stimulate expression of MHC II on tumor cells, thereby promoting CD4(+) T cell activation. In this study, N2a neuroblastoma cells were stably transfected with CIITA. N2aCIITA cells displayed increased expression of MHC I, MHC II and invariant chain; CD80 and CD86 were expressed by neither the parental N2a cells nor by the N2aCIITA cells. All mice injected with N2aCIITA cells developed tumors. Furthermore, no increase in the numbers of T cells, natural killer cells, macrophages, or eosinophils was observed in the spleens or tumors of mice injected with N2aCIITA cells, compared to tissues from mice injected with the parental N2a cells. This absence of an anti-tumor immune response despite MHC II expression is likely due to the presence of invariant chain, in support of the MHCII(+)/Ii(-) paradigm.

  20. In vivo bioluminescence and reflectance imaging of multiple organs in bioluminescence reporter mice by bundled-fiber-coupled microscopy

    PubMed Central

    Ando, Yoriko; Sakurai, Takashi; Koida, Kowa; Tei, Hajime; Hida, Akiko; Nakao, Kazuki; Natsume, Mistuo; Numano, Rika

    2016-01-01

    Bioluminescence imaging (BLI) is used in biomedical research to monitor biological processes within living organisms. Recently, fiber bundles with high transmittance and density have been developed to detect low light with high resolution. Therefore, we have developed a bundled-fiber-coupled microscope with a highly sensitive cooled-CCD camera that enables the BLI of organs within the mouse body. This is the first report of in vivo BLI of the brain and multiple organs in luciferase-reporter mice using bundled-fiber optics. With reflectance imaging, the structures of blood vessels and organs can be seen clearly with light illumination, and it allowed identification of the structural details of bioluminescence images. This technique can also be applied to clinical diagnostics in a low invasive manner. PMID:27231601

  1. In vivo bioluminescence and reflectance imaging of multiple organs in bioluminescence reporter mice by bundled-fiber-coupled microscopy.

    PubMed

    Ando, Yoriko; Sakurai, Takashi; Koida, Kowa; Tei, Hajime; Hida, Akiko; Nakao, Kazuki; Natsume, Mistuo; Numano, Rika

    2016-03-01

    Bioluminescence imaging (BLI) is used in biomedical research to monitor biological processes within living organisms. Recently, fiber bundles with high transmittance and density have been developed to detect low light with high resolution. Therefore, we have developed a bundled-fiber-coupled microscope with a highly sensitive cooled-CCD camera that enables the BLI of organs within the mouse body. This is the first report of in vivo BLI of the brain and multiple organs in luciferase-reporter mice using bundled-fiber optics. With reflectance imaging, the structures of blood vessels and organs can be seen clearly with light illumination, and it allowed identification of the structural details of bioluminescence images. This technique can also be applied to clinical diagnostics in a low invasive manner.

  2. Histone H2A significantly enhances in vitro DNA transfection.

    PubMed Central

    Balicki, D.; Beutler, E.

    1997-01-01

    BACKGROUND: Gene transfer is a potential treatment modality of genetic disease. Efficient, practical methods of DNA transfection are currently under investigation. MATERIALS AND METHODS: A beta-galactosidase reporter plasmid interacted electrostatically with histones, poly-L-Lys, poly-L-Arg, and a combination of poly-L-Lys and poly-L-Arg. This complex was then used to transfect COS-7 cells. beta-galactosidase activity was quantified and used to compare the efficiency of gene transfection in vitro. A comparison was also made of DNA transfection with the most active histone subclass, i.e., histone H2A, in the absence and presence of an anionic liposome. RESULTS: There was a marked increase in DNA transfection in the presence of histone H2A when compared with the control, whereas each of the other histones and polycations showed little, if any, effect. The extent of activation depends strongly on the DNA/histone ratio and is also a function of the molarity of the final Tris-acetate, pH 8, solution. The anionic liposomes used demonstrated an inhibitory effect. CONCLUSIONS: Histone H2A significantly enhances in vitro DNA transfection whereas other histones and anionic liposomes do not. A study of the difference between histone H2A and other histone subclasses may serve to clarify some of the mechanisms and the essential components of efficient gene delivery. PMID:9407553

  3. Synchronization of circadian bioluminescence as a group-foraging strategy in cave glowworms.

    PubMed

    Maynard, Andrew J; Merritt, David J

    2013-07-01

    Flies of the genus Arachnocampa are sit-and-lure predators that use bioluminescence to attract flying prey to their silk webs. Some species are most common in rainforest habitat and others inhabit both caves and rainforest. We have studied the circadian regulation of bioluminescence in two species: one found in subtropical rainforest with no known cave populations and the other found in temperate rainforest with large populations in limestone caves. The rainforest species is typical of most nocturnal animals in that individuals are entrained by the light:dark (LD) cycle to be active at night; in this case, their propensity to bioluminesce is greatest at night. The dual-habitat species shows an opposite phase response to the same entrainment; its bioluminescence propensity rhythm is entrained by LD exposure to peak during the day. Nevertheless, in LD environments, individuals do not bioluminesce during the day because ambient light inhibits their bioluminescence (negative masking), pushing bioluminescence into the dark period. This unusual and unexpected phenomenon could be related to their association with caves and has been suggested to be an adaptation of the circadian system that promotes synchronization of a colony's output of bioluminescence. Here, we use controlled laboratory experiments to show that individuals do synchronize their bioluminescence rhythms when in visual contact with each other. Entrainment of the bioluminescence rhythm to the biological photophase causes colony-wide synchronization, creating a daily sinusoidal rhythm of the intensity of bioluminescence in the many thousands of individuals making up a colony. This synchronization could provide a group-foraging advantage, allowing the colony to glow most brightly when the prey are most likely to be active.

  4. Radiofrequency transmission line for bioluminescent Vibrio sp. irradiation

    NASA Astrophysics Data System (ADS)

    Nassisi, V.; Alifano, P.; Talà, A.; Velardi, L.

    2012-07-01

    We present the study and the analyses of a transmission line for radiofrequency (RF) irradiation of bacteria belonging to Vibrio harveyi-related strain PS1, a bioluminescent bacterium living in symbiosis with many marine organisms. The bioluminescence represents a new biologic indicator which is useful for studying the behaviour of living samples in the presence of RF waves due to the modern communication systems. A suitable transmission line, used as an irradiating cell and tested up to the maximum frequency used by the global system for mobile communications and universal mobile telecommunications system transmissions, was characterized. In this experiment, the RF voltage applied to the transmission line was 1 V. Due to short dimensions of the line and the applied high frequencies, standing waves were produced in addition to progressing waves and the electric field strength varies particularly along the longitudinal direction. The magnetic field map was not strongly linked to the electric one due to the presence of standing waves and of the outgoing irradiation. RF fields were measured by two homemade suitable probes able to diagnostic fields of high frequency. The field measurements were performed without any specimens inside the line. Being our sample made of living matter, the real field was modified and its value was estimated by a simulation code. The bioluminescence experiments were performed only at 900 MHz for two different measured electric fields, 53 and 140 V/m. The light emission was measured right from the beginning and after 7 and 25 h. Under RF irradiation, we found that the bioluminescence activity decreased. Compared with the control sample, the diminution was 6.8% and 44% after 7 and 25 h of irradiation, respectively, both with the low or high field. No changes of the survival factor for all the samples were observed. Besides, to understand the emission processes, we operated the deconvolution of the spectra by two Gaussian curves. The Gaussian

  5. An adaptive regularization parameter choice strategy for multispectral bioluminescence tomography

    SciTech Connect

    Feng Jinchao; Qin Chenghu; Jia Kebin; Han Dong; Liu Kai; Zhu Shouping; Yang Xin; Tian Jie

    2011-11-15

    Purpose: Bioluminescence tomography (BLT) provides an effective tool for monitoring physiological and pathological activities in vivo. However, the measured data in bioluminescence imaging are corrupted by noise. Therefore, regularization methods are commonly used to find a regularized solution. Nevertheless, for the quality of the reconstructed bioluminescent source obtained by regularization methods, the choice of the regularization parameters is crucial. To date, the selection of regularization parameters remains challenging. With regards to the above problems, the authors proposed a BLT reconstruction algorithm with an adaptive parameter choice rule. Methods: The proposed reconstruction algorithm uses a diffusion equation for modeling the bioluminescent photon transport. The diffusion equation is solved with a finite element method. Computed tomography (CT) images provide anatomical information regarding the geometry of the small animal and its internal organs. To reduce the ill-posedness of BLT, spectral information and the optimal permissible source region are employed. Then, the relationship between the unknown source distribution and multiview and multispectral boundary measurements is established based on the finite element method and the optimal permissible source region. Since the measured data are noisy, the BLT reconstruction is formulated as l{sub 2} data fidelity and a general regularization term. When choosing the regularization parameters for BLT, an efficient model function approach is proposed, which does not require knowledge of the noise level. This approach only requests the computation of the residual and regularized solution norm. With this knowledge, we construct the model function to approximate the objective function, and the regularization parameter is updated iteratively. Results: First, the micro-CT based mouse phantom was used for simulation verification. Simulation experiments were used to illustrate why multispectral data were used

  6. Bioluminescent bioreporter integrated circuit devices and methods for detecting ammonia

    DOEpatents

    Simpson, Michael L [Knoxville, TN; Paulus, Michael J [Knoxville, TN; Sayler, Gary S [Blaine, TN; Applegate, Bruce M [West Lafayette, IN; Ripp, Steven A [Knoxville, TN

    2007-04-24

    Monolithic bioelectronic devices for the detection of ammonia includes a microorganism that metabolizes ammonia and which harbors a lux gene fused with a heterologous promoter gene stably incorporated into the chromosome of the microorganism and an Optical Application Specific Integrated Circuit (OASIC). The microorganism is generally a bacterium.

  7. Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.

    PubMed

    Liu, Lin-Lin; Zhao, Hui; Ma, Teng-Fei; Ge, Fei; Chen, Ce-Shi; Zhang, Ya-Ping

    2015-01-01

    Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct), and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2) expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.

  8. Stability boundaries and sufficient stability conditions for stably stratified, monotonic shear flows

    NASA Astrophysics Data System (ADS)

    Hirota, Makoto; Morrison, Philip J.

    2016-05-01

    Linear stability of inviscid, parallel, and stably stratified shear flow is studied under the assumption of smooth strictly monotonic profiles of shear flow and density, so that the local Richardson number is positive everywhere. The marginally unstable modes are systematically found by solving a one-parameter family of regular Sturm-Liouville problems, which can determine the stability boundaries more efficiently than solving the Taylor-Goldstein equation directly. By arguing for the non-existence of a marginally unstable mode, we derive new sufficient conditions for stability, which generalize the Rayleigh-Fjørtoft criterion for unstratified shear flows.

  9. Second moment closure modeling for rotating stably stratified turbulent shear flow

    NASA Astrophysics Data System (ADS)

    Ji, Minsuk

    The general linear second moment closure (SMC) turbulence model is considered for flows subjected to buoyancy and rotation. Model response to external forces are analyzed with the aid of structural equilibrium analysis. A closed form equilibrium solution for the anisotropy tensor bij, dispersion tensor Kij, dimensionless scalar variance q2 /k(S/Stheta )2, and the ratio of mean to turbulent time scale epsilon/ Sk is obtained. The variable of particular interest to bifurcation analysis, epsilon/Sk is shown as a function of the parameters characterizing the body forces: O/S (the ratio of the rotation rate to the mean shear rate) for rotation and Rig (the gradient Richardson number) for buoyancy; it determines the bifurcation surface in the epsilon/Sk-O/S-Rig space. It is shown, with the use of the closed form solution, that the conventional general linear models do not have a real and stable equilibrium solution when rotational and buoyant forces of certain magnitudes are simultaneously imposed on the flow. When this occurs, time integration of the turbulence model results in a diverging solution. A new model is proposed that removes this unphysical behavior. It ensures the existence of stable, real solutions for all combinations of rotation and buoyancy. Further improvements to the model are made through bifurcation analysis. Model constants are adjusted such that the model's bifurcation characteristics are in agreement with the physically observed onset of turbulence stabilization due to stable stratification. Experimental data and numerical simulation results for stably stratified homogeneous shear flow suggest the critical gradient Richardson number of Ricrg = 0.25, and the new model is able to predict it correctly. In connection with the bifurcation analysis of SMC models, rapid distortion theory (RDT) of turbulence is applied to rotating, stably stratified shear flow to provide the stability characteristics of such flows. It is shown that the RDT predictions are

  10. Inverted floor wind-tunnel simulation of stably stratified atmospheric boundary layer flow

    NASA Astrophysics Data System (ADS)

    Grainger, Clive; Meroney, Robert N.

    Most of the critical transport processes in the atmosphere are dominated by density stratification; hence, physical modeling facilities which neglect the important contributions of buoyancy are limited to the examination of high winds or those brief moments after sunrise or before sunset when the atmosphere is nominally neutrally stratified. Large new facilities constructed specifically to simulate the atmosphere offer new opportunities to study the physics of mixing processes dominated by stratification. A novel arrangement to simulate stably stratified atmospheric boundary layer flows in large wind tunnels using distributed electrical heaters and an inverted ground plane to simulate nighttime inversions is described, together with initial measurements.

  11. Using bioluminescent biosensors for hazard analysis and critical control point (HACCP) in wastewater control.

    PubMed

    Valat, C; Champiat, D; Degorce-Dumas, J R; Thomas, O

    2004-01-01

    Starting from a new approach for water pollution control and wastewater treatment plant management, the hazard analysis and critical control point (HACCP) quality concept, the interest for the development of new rapid and sensitive methods such as bioluminescence-based methods is evident. After an introduction of the HACCP procedure, a bibliographic study of the bioluminescence potentiality is presented and discussed.

  12. Evaluation of ATP bioluminescence assays for potential use in a hospital setting.

    PubMed

    Aiken, Zoie A; Wilson, Michael; Pratten, Jonathan

    2011-05-01

    ATP bioluminescence is being applied in hospitals to measure surface contamination. We compared commercial luminometers for detecting the number Staphylococcus aureus associated with surfaces. The data showed that the ATP bioluminescence methods tested were not robust enough to generate quantitative data on bacterial numbers, especially at low concentrations.

  13. In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase

    SciTech Connect

    Liu Hua; Luan Fang; Ju Ying; Shen Hongyu; Gao Lifen; Wang Xiaoyan; Liu Suxia; Zhang Lining; Sun Wensheng; Ma Chunhong . E-mail: machunhong@sdu.edu.cn

    2007-04-06

    The preS2 domain is the minimal functional unit of transcription activators that is encoded by the Hepatitis B virus (HBV) surface (S) gene. It is present in more than one-third of the HBV-integrates in HBV induced hepatocarcinoma (HCC). To further understand the functional role of PreS2 in hepatocytes, a PreS2 expression plasmid, pcS2, was constructed and stably transfected into HepG2 cells. We conducted growth curve and colony-forming assays to study the impact of PreS2 expression on cell proliferation. Cells transfected with PreS2 proliferated more rapidly and formed colonies in soft agar. PreS2 expressing cells also induced upregulation of human telomerase reverse transcriptase (hTERT) and telomerase activation by RT-PCR and the modified TRAP assay. Blocking expression of hTERT with antisense oligonuleotide reversed the growth rate in cells stably transfected with PreS2. Our data suggest that PreS2 may increase the malignant transformation of human HCC cell line HepG2 by upregulating hTERT and inducing telomerase activation.

  14. Bioluminescent monitoring of in vivo colonization and clearance dynamics by light-emitting bacteria.

    PubMed

    Wiles, Siouxsie; Robertson, Brian D; Frankel, Gad; Kerton, Angela

    2009-01-01

    Bioluminescence is an excellent reporter system for analysing bacterial colonization and clearance dynamics in vivo. Many bacterial species have been rendered bioluminescent, allowing the sensitive detection of bacterial burden and metabolic activity in real-time and in situ in living animals. In this chapter we describe the protocols for characterizing in vivo infection models using bioluminescent bacteria: from real-time imaging in living animals by bioluminescence imaging (BLI) to ex vivo BLI of harvested organs and tissues and, finally, to quantification of bacterial numbers in organ and tissue homogenates by luminometry and viable counts. While the lux operon from Photorhabdus luminescens is ideally suited for use in such models, there may be times when alternative luciferases, such as those from the firefly (luc) or marine copepods (Gluc), may be more appropriate. Here we describe the protocols required to monitor colonization and clearance dynamics using bioluminescent bacteria that are lux-, luc-, or Gluc-positive.

  15. Femtosecond cellular transfection using a non-diffracting beam

    NASA Astrophysics Data System (ADS)

    Tsampoula, X.; Garcés-Chávez, V.; Comrie, M.; Stevenson, D. J.; Agate, B.; Brown, C. T. A.; Gunn-Moore, F.; Dholakia, K.

    2008-02-01

    Efficient DNA delivery into single living cells would be a very powerful capability for cell biologists for elucidating basic cellular functions but also in other fields such as applied drug discovery and gene therapy. The ability to gently permeate the cell membrane and introduce foreign DNA with the assistance of lasers is a powerful methodology but requires exact focusing due to the required two-photon power density. Here, we demonstrate a laser-mediated delivery method of the red fluorescent protein DS-RED into Chinese hamster Ovary (CHO) cells. We used an elongated beam of light created by a Bessel beam (BB) which obviates the need to locate precisely the cell membrane, permitting two-photon excitation along a line leading to cell transfection. Assuming a threshold for transfection of 20%, the BB gives us transfection over twenty times the axial distance compared to the Gaussian beam of equivalent core diameter. In addition, by exploiting the BB property of reconstruction, we demonstrate successful transfection of CHO cells which involves the BB passing through an obstructive layer and re forming itself prior to reaching the cell membrane. In the light of this exciting result, one can envisage the possibility of achieving transfection through multiple cell monolayer planes and tissues using this novel light field, eliminating this way the stringent requirements for tight focusing.

  16. Application of ATP-based bioluminescence for bioaerosol quantification: effect of sampling method.

    PubMed

    Han, Taewon; Wren, Melody; DuBois, Kelsey; Therkorn, Jennifer; Mainelis, Gediminas

    2015-12-01

    An adenosine triphosphate (ATP)-based bioluminescence has potential to offer a quick and affordable method for quantifying bioaerosol samples. Here we report on our investigation into how different bioaerosol aerosolization parameters and sampling methods affect bioluminescence output per bacterium, and implications of that effect for bioaerosol research. Bacillus atrophaeus and Pseudomonas fluorescens bacteria were aerosolized by using a Collison nebulizer (BGI Inc., Waltham, MA) with a glass or polycarbonate jar and then collected for 15 and 60 min with: (1) Button Aerosol Sampler (SKC Inc., Eighty Four, PA) with polycarbonate, PTFE, and cellulose nitrate filters, (2) BioSampler (SKC Inc.) with 5 and 20 mL of collection liquid, and (3) our newly developed Electrostatic Precipitator with Superhydrophobic Surface (EPSS). For all aerosolization and sampling parameters we compared the ATP bioluminescence output per bacterium relative to that before aerosolization and sampling. In addition, we also determined the ATP reagent storage and preparation conditions that that do not affect the bioluminescence signal intensity. Our results show that aerosolization by a Collison nebulizer with a polycarbonate jar yields higher bioluminescence output per bacterium compared to the glass jar. Interestingly enough, the bioluminescence output by P. fluorescens increased substantially after its aerosolization compared to the fresh liquid suspension. For both test microorganisms, the bioluminescence intensity per bacterium after sampling was significantly lower than that before sampling suggesting negative effect of sampling stress on bioluminescence output. The decrease in bioluminescence intensity was more pronounces for longer sampling times and significantly and substantially depended on the sampling method. Among the investigated method, the EPSS was the least injurious for both microorganisms and sampling times. While the ATP-based bioluminescence offers a quick bioaerosol

  17. Application of ATP-based bioluminescence for bioaerosol quantification: effect of sampling method

    PubMed Central

    Han, Taewon; Wren, Melody; DuBois, Kelsey; Therkorn, Jennifer; Mainelis, Gediminas

    2015-01-01

    An adenosine triphosphate (ATP)-based bioluminescence has potential to offer a quick and affordable method for quantifying bioaerosol samples. Here we report on our investigation into how different bioaerosol aerosolization parameters and sampling methods affect bioluminescence output per bacterium, and implications of that effect for bioaerosol research. Bacillus atrophaeus and Pseudomonas fluorescens bacteria were aerosolized by using a Collison nebulizer (BGI Inc., Waltham, MA) with a glass or polycarbonate jar and then collected for 15 and 60 min with: (1) Button Aerosol Sampler (SKC Inc., Eighty Four, PA) with polycarbonate, PTFE, and cellulose nitrate filters, (2) BioSampler (SKC Inc.) with 5 and 20 mL of collection liquid, and (3) our newly developed Electrostatic Precipitator with Superhydrophobic Surface (EPSS). For all aerosolization and sampling parameters we compared the ATP bioluminescence output per bacterium relative to that before aerosolization and sampling. In addition, we also determined the ATP reagent storage and preparation conditions that that do not affect the bioluminescence signal intensity. Our results show that aerosolization by a Collison nebulizer with a polycarbonate jar yields higher bioluminescence output per bacterium compared to the glass jar. Interestingly enough, the bioluminescence output by P. fluorescens increased substantially after its aerosolization compared to the fresh liquid suspension. For both test microorganisms, the bioluminescence intensity per bacterium after sampling was significantly lower than that before sampling suggesting negative effect of sampling stress on bioluminescence output. The decrease in bioluminescence intensity was more pronounces for longer sampling times and significantly and substantially depended on the sampling method. Among the investigated method, the EPSS was the least injurious for both microorganisms and sampling times. While the ATP-based bioluminescence offers a quick bioaerosol

  18. A Sweeping based Kinematic Simulation for the Stably Stratified Surface Layer

    NASA Astrophysics Data System (ADS)

    Ghate, Aditya; Lele, Sanjiva

    2014-11-01

    A Kinematic Simulation (KS) for a statistically stationary and stably stratified surface layer is proposed. The Fourier coefficients are obtained by numerically solving the linearized NS equations with Boussinesq approximation in spectral space, under the assumption of ``rapid'' deformation (RDT) due to combined shear and stratification. The linearization of RDT, which is unrealistic for the surface layer, is rectified using Mann's (JFM, 1994) idea of wavenumber dependent eddy lifetime. The input parameters required by the KS are estimated using either Monin-Obukhov theory, or an appropriate Second Moment Closure. In order to overcome the frozen turbulence hypothesis made in the Mann model, we incorporate inter-scale ``sweeping'' of eddies following the ideas of Fung et al. (JFM, 1992), along with temporal decorrelation associated with the natural eddy time scale. The solenoidal velocity field generated by the KS allows inclusion of a wide range of scales with correct space-time correlations, making it ideal to investigate particle dispersion in a stably stratified environment, and can also serve as inflow for the study of Wind Farm-PBL interactions. The effect of varying Obukhov length will be discussed by analyzing the frozen Eulerian spectra and Lagrangian particle dispersion.

  19. Archetype JC virus efficiently propagates in kidney-derived cells stably expressing HIV-1 Tat.

    PubMed

    Nukuzuma, Souichi; Kameoka, Masanori; Sugiura, Shigeki; Nakamichi, Kazuo; Nukuzuma, Chiyoko; Miyoshi, Isao; Takegami, Tsutomu

    2009-11-01

    Pathogenic JCV with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease, in the brains of immunocompromised patients. On the other hand, archetype JCV persistently infecting the kidney is thought to be converted to PML-type virus during JCV replication in the infected host under immunosuppressed conditions. In addition, Tat protein, encoded by HIV-1, markedly enhances the expression of a reporter gene under control of the JCV late promoter. In order to examine the influence of Tat on JCV propagation, we used kidney-derived COS-7 cells, which only permit archetype JCV, and established COS-tat cells, which express HIV-1 Tat stably. We found that the extent of archetype JCV propagation in COS-tat cells is significantly greater than in COS-7 cells. On the other hand, COS-7 cells express SV40 T antigen, which is a strong stimulator of archetype JCV replication. The expression of SV40 T antigen was enhanced by HIV-1 Tat slightly according to real-time RT-PCR, this was not closely related to JCV replication in COS-tat cells. The efficiency of JCV propagation depended on the extent of expression of functional Tat. To our knowledge, this is the first report of increased production of archetype JCV in a culture system using cell lines stably expressing HIV-1 Tat. We propose here that COS-tat cells are a useful tool for studying the role of Tat in archetype JCV replication in the development of PML.

  20. Effect of thermal boundary condition on wall-bounded, stably-stratified turbulence

    NASA Astrophysics Data System (ADS)

    Flores, Oscar; Garcia-Villalba, Manuel

    2012-11-01

    The dynamics of stably stratified wall-bounded turbulent flows are of great importance for many engineering and geophysical problems. In some cases, like the stably stratified atmospheric boundary layer, it is unclear which is the most appropriate thermal boundary condition, i.e. constant temperature or constant flux at the ground. Here, we analyze the effect that this boundary condition has on the dynamics of turbulent motions in the near-wall region in the case of strong stable stratification. Two Direct Numerical Simulations of turbulent channels will be used, at Reτ =uτ h / ν = 560 and Riτ = Δρgh /ρ0uτ2 = 600 - 900 , which are described in detail in Flores & Riley (2011, Boundary-Layer Meteorol) and Garcia-Villalba & del Alamo (2011, Phys.Fluids). For this range of Reynolds and Richardson numbers, the near-wall region is intermittent, with patches of laminar flow embedded in the otherwise turbulent flow. It is in this regime where the differences between the constant temperature and the constant flux boundary conditions are expected to be larger, with the thermal boundary condition affecting how the local relaminarization of the flow takes place. This research has been supported by ARO, NSF and the German Research Foundation.

  1. A Numerical Model for Magnetohydrodynamic Waves in a Stably-Stratified Layer in Earth's Core

    NASA Astrophysics Data System (ADS)

    Knezek, N. R.; Buffett, B. A.

    2015-12-01

    A numerical model for magnetohydrodynamic waves in a thin shell is developed and applied to study the effect of a stably-stratified layer in Earth's core on geomagnetic secular variation. The model employs a spherical coordinate system with finite differences in r and θ and Fourier decomposition in Φ. The model is linearized assuming a background azimuthal velocity field UΦ(r,θ) and an arbitrary background magnetic field Br,θ,Φ(r,θ). The Boussinesq approximation is employed and the buoyancy forces are prescribed in terms of a spatially variable Brunt-Vaisala frequency N(r,θ). The equations are cast into a sparse generalized eigenvalue problem by assuming solutions of the form uj,bj,p=CjeimΦ+λt and eigenmodes are found. Good agreement is obtained with previous approximate analytical solutions for zonal (m=0) magnetic-Archimedes-Coriolis (MAC) waves (e.g. Braginsky, 1993), global magnetic-Rossby (m>0) waves (e.g. Braginsky, 1998), and equatorially-trapped magnetic-Rossby waves (e.g. Bergman, 1993). This model is employed to study the origins of the fast equatorial waves observed by Chulliat et al. (2015) in recent high-resolution magnetic field models to constrain plausible properties of the stably-stratified layer and core-surface magnetic field.

  2. Gene transfection of HEK cells on supermacroporous polyacrylamide monoliths: a comparison of transient and stable recombinant protein expression in perfusion culture.

    PubMed

    Cheeks, Matthew C; Edwards, Alexander D; Arnot, Christopher J; Slater, Nigel K H

    2009-12-31

    Transient and continuous recombinant protein expression by HEK cells was evaluated in a perfused monolithic bioreactor. Highly porous synthetic cryogel scaffolds (10 ml bed volume) were characterised by scanning electron microscopy and tested as cell substrates. Efficient seeding was achieved (94% inoculum retained, with 91-95% viability). Metabolite monitoring indicated continuous cell growth, and endpoint cell density was estimated by genomic DNA quantification to be 5.2 x 10(8), 1.1 x 10(9) and 3.5 x 10(10) at day 10, 14 and 18. Culture of stably transfected cells allowed continuous production of the Drosophila cytokine Spätzle by the bioreactor at the same rate as in monolayer culture (total 1.2mg at day 18) and this protein was active. In transient transfection experiments more protein was produced per cell compared with monolayer culture. Confocal microscopy confirmed homogenous GFP expression after transient transfection within the bioreactor. Monolithic bioreactors are thus a flexible and powerful tool for manufacturing recombinant proteins.

  3. Viral susceptibility, transfection and growth of SPB--a fish neural progenitor cell line from the brain of snubnose pompano, Trachinotus blochii (Lacépède).

    PubMed

    Wen, C-M; Ku, C-C; Wang, C-S

    2013-07-01

    This study investigates the susceptibilities of the SPB cell line to fish viruses including giant seaperch iridovirus (GSIV-K1), red sea bream iridovirus (RSIV-Ku), grouper nervous necrosis virus (GNNV-K1), chum salmon reovirus (CSV) and eel herpesvirus (HVA). GSIV-K1, RSIV-Ku and CSV replicated well in SPB cells, with a significant cytopathic effect and virus production. However, the cells were HVA and GNNV refractory. To examine the ability of SPB cells to stably express foreign protein, expression vectors encoding GNNV B1 and B2 fused to enhanced green fluorescent protein (EGFP) and GSIV ORF35L fused to DsRed were constructed and introduced by transfection into SPB cells. Stable transfectants displayed different morphologies compared with SPB and with each other. EGFP-B1 was predominantly localized in the nuclei, EFPF-B2 was distributed throughout the cytoplasm and nucleus, and granular 35L-DsRed was localized with secreted vesicles. The expression of EFPF-B2 in SPB cells produced blebs on the surface, but the cells showing stable expression of EGFP, EGFP-B1 or 35L-DsRed showed normal morphologies. Results show the SPB cells and the transfected cells grow well at temperatures between 20 and 35 °C and with serum-dependent growth. SPB cells are suitable for studies on foreign protein expression and virology.

  4. Synthesis of linear polyethylenimine derivatives for DNA transfection.

    PubMed

    Brissault, Blandine; Kichler, Antoine; Guis, Christine; Leborgne, Christian; Danos, Olivier; Cheradame, Hervé

    2003-01-01

    A series of linear polymers containing varying amounts of ethylenimine or N-propylethylenimine units were synthesized by hydrolysis and/or reduction of polyethyloxazolines. The pK(a)s of the polyamines were determined potentiometrically. Gel mobility shift assay showed that the efficiency of DNA complexation was related to the fraction of amino groups that are protonated at neutral pH. The effects of cationic charge density and molar weight of the polymers on the transfection efficiency were evaluated on HepG2 cells. The results obtained with different copolymers show that the transfection efficiency primarily depends on the fraction of ethylenimine units included in the polymer albeit the molar weight is also of importance. On the basis of the results obtained with poly(N-propylethylenimines), we also demonstrate that the high transfection efficiency of polyethylenimines does not solely rely on their capacity to capture protons which are transferred into the endo-lysosomes during acidification.

  5. In trans promoter activation by enhancers in transient transfection.

    PubMed

    Smirnov, N A; Akopov, S B; Didych, D A; Nikolaev, L G

    2017-03-01

    Earlier, it was reported that the strong cytomegalovirus enhancer can activate the cytomegalovirus promoter in trans, i.e. as a separate plasmid co-transfected with a promoter-reporter gene construct. Here we demonstrate that the ability of enhancers to activate promoters in trans in transient transfection experiments is a property of not only viral regulatory elements but also of various genomic enhancers and promoters. Enhancer-promoter activation in trans is promoter- and cell type-specific, and accompanied by physical interaction between promoter and enhancer as revealed by chromosome conformation capture assays. Thus, promoter activation in transient co-transfection of promoters and enhancers shares a number of important traits with long-distance promoter activation by enhancers in living cells and may therefore serve as a model of this fundamental cellular process.

  6. 96-well electroporation method for transfection of mammalian central neurons.

    PubMed

    Buchser, William J; Pardinas, Jose R; Shi, Yan; Bixby, John L; Lemmon, Vance P

    2006-11-01

    Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to obtain better efficiencies. We have developed an efficient method for the transfection of cerebellar granule neurons and hippocampal neurons with standard plasmid vectors. Using 96-well electroporation plates, square-wave pulses can introduce 96 different plasmids into neurons in a single step. The procedure results in greater than 20% transfection efficiencies and requires only simple solutions of nominal cost. In addition to enabling the rapid optimization of experimental protocols with multiple parameters, this procedure enables the use of high content screening methods to characterize neuronal phenotypes.

  7. 96-Well electroporation method for transfection of mammalian central neurons

    PubMed Central

    Buchser, William J.; Pardinas, Jose R.; Shi, Yan; Bixby, John L.; Lemmon, Vance P.

    2008-01-01

    Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to obtain better efficiencies. We have developed an efficient method for the transfection of cerebellar granule neurons and hippocampal neurons with standard plasmid vectors. Using 96-well electroporation plates, square-wave pulses can introduce 96 different plasmids into neurons in a single step. The procedure results in greater than 20% transfection efficiencies and requires only simple solutions of nominal cost. In addition to enabling the rapid optimization of experimental protocols with multiple parameters, this procedure enables the use of high content screening methods to characterize neuronal phenotypes. PMID:17140120

  8. Bioluminescence tomography guided radiation therapy for preclinical research

    PubMed Central

    Zhang, Bin; Wang, Ken Kang-Hsin; Yu, Jingjing; Eslami, Sohrab; Iordachita, Iulian; Reyes, Juvenal; Malek, Reem; Tran, Phuoc T.; Patterson, Michael S.; Wong, John W.

    2015-01-01

    Purpose In pre-clinical radiation research, it is challenging to localize soft tissue targets based on cone beam computed tomography (CBCT)-guidance. As a more effective method to localize soft tissue targets, we developed an online bioluminescence tomography (BLT) system for the small animal radiation research platform (SARRP). We demonstrated BLT-guided radiotherapy and validated targeting accuracy, based on a newly developed reconstruction algorithm. Methods and Materials The BLT system was designed to dock onto the SARRP for image acquisition and to be detached before radiation delivery. A 3-mirror system was devised to reflect the bioluminescence emitted from the subject to a stationary CCD camera. Multispectral BLT and the incomplete variables truncated conjugate gradient method with a permissible region shrinking strategy were employed as the optimization scheme to reconstruct bioluminescent source distributions. To validate BLT targeting accuracy, a small cylindrical light source with high CBCT contrast was placed in a phantom and also in the abdomen of a mouse carcass. The center of mass (CoM) of the source was recovered from BLT and used to guide radiation delivery. The accuracy of the BLT-guided targeting was validated with films and compared with the CBCT-guided delivery. In vivo experiments were conducted to demonstrate the BLT localization capability for various source geometries. Results Online BLT was able to recover the CoM of the imbedded light source with an average accuracy of 1 mm compared to CBCT localization. The difference between the BLT- and CBCT-guided irradiation shown on the films was consistent with the source localization revealed in the BLT and CBCT images. The in vivo results demonstrated that our BLT system could potentially be applied for multiple targets and tumors. Conclusions The online BLT/CBCT/SARRP system provides an effective solution for soft tissue targeting, particularly for small, non-palpable, or orthotopic tumor

  9. Mutagenesis and Characterization Studies to Develop Novel Bioluminescent Systems

    DTIC Science & Technology

    2010-05-12

    Doyle, S.M. Burns , C.H. Contag, In vivo bioluminescence imaging for integrated studies of infection, Cellular Microbiology 6 (2004) 303-317. [14] C.M...37 °C in LB broth supplemented with 100 g/ml ampicillin to mid log phase (A600 = 0.6-0.9), transferred to a 22 °C incubator and, after...13230. (30) Branchini, B. R., Magyar, R. A., Murtiashaw, M. H., and Portier, N. C. (2001) The role of active site residue arginine 218 in firefly

  10. Luciferase-dependent oxygen consumption by bioluminescent vibrios.

    PubMed Central

    Makemson, J C

    1986-01-01

    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. PMID:3944057

  11. Antioxidant assay using genetically engineered bioluminescent Escherichia coli

    NASA Astrophysics Data System (ADS)

    Bartolome, Amelita; Macalino, Bernadette; Pastoral, Ian Lemuel; Sevilla, Fortunato, III

    2006-02-01

    A new antioxidant activity assay based on the reactive oxygen species (ROS)-inducible bacterial strain (E. coli DPD2511) is described. The strain harbors the plasmid pKatG::luxCDABE and responds to hydrogen peroxide treatment by increasing light emission at 490 nm. Antioxidant capacity is evaluated through the ability of an agent to inhibit the hydrogen peroxide-induced bioluminescence of E. coli DPD2511. Applicability of the developed assay in detecting levels of antioxidants in various aqueous plant extracts is demonstrated. The assay was validated against 2,2-diphenylpicrylhydrazyl (DPPH) assay, a known antioxidant assay.

  12. 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.

  13. Bioluminescence monitor and method for enzymatic determinations. [Patents

    SciTech Connect

    Bostick, W.D.; Denton, M.S.; Dinsmore, S.R.

    1981-04-28

    An on-line, nonreferenced apparatus for measuring the concentration of a biomarker species in authentic biological samples in solution comprises conduit means for conducting said sample solution from a source of said solution, stream diversion means disposed within the conduit for diverting a predetermined amount of said sample for analysis, means for introducing and independently regulating the flow of one or more reactants disposed in fluid communication with said diverted stream, incubating means within the diverted stream for reacting said reactants and biomarkers to produce a bioluminescence emission, and means disposed within the diverted stream for monitoring said emission intensity which is correlatable to said biomarker concentration.

  14. Bioluminescence Tomography: Biomedical Background, Mathematical Theory, and Numerical Approximation 1)

    PubMed Central

    Han, Weimin; Wang, Ge

    2010-01-01

    Over the last couple of years molecular imaging has been rapidly developed to study physiological and pathological processes in vivo at the cellular and molecular levels. Among molecular imaging modalities, optical imaging stands out for its unique advantages, especially performance and cost-effectiveness. Bioluminescence tomography (BLT) is an emerging optical imaging mode with promising biomedical advantages. In this survey paper, we explain the biomedical significance of BLT, summarize theoretical results on the analysis and numerical solution of a diffusion based BLT model, and comment on a few extensions for the study of BLT. PMID:20617105

  15. Experimental Study on Bioluminescence Tomography with Multimodality Fusion

    PubMed Central

    Lv, Yujie; Tian, Jie; Cong, Wenxiang; Wang, Ge

    2007-01-01

    To verify the influence of a priori information on the nonuniqueness problem of bioluminescence tomography (BLT), the multimodality imaging fusion based BLT experiment is performed by multiview noncontact detection mode, which incorporates the anatomical information obtained by the microCT scanner and the background optical properties based on diffuse reflectance measurements. In the reconstruction procedure, the utilization of adaptive finite element methods (FEMs) and a priori permissible source region refines the reconstructed results and improves numerical robustness and efficiency. The comparison between the absence and employment of a priori information shows that multimodality imaging fusion is essential to quantitative BLT reconstruction. PMID:18256736

  16. Quantification of bioluminescence from the surface to the deep sea demonstrates its predominance as an ecological trait.

    PubMed

    Martini, Séverine; Haddock, Steven H D

    2017-04-04

    The capability of animals to emit light, called bioluminescence, is considered to be a major factor in ecological interactions. Because it occurs across diverse taxa, measurements of bioluminescence can be powerful to detect and quantify organisms in the ocean. In this study, 17 years of video observations were recorded by remotely operated vehicles during surveys off the California Coast, from the surface down to 3,900 m depth. More than 350,000 observations are classified for their bioluminescence capability based on literature descriptions. The organisms represented 553 phylogenetic concepts (species, genera or families, at the most precise taxonomic level defined from the images), distributed within 13 broader taxonomic categories. The importance of bioluminescent marine taxa is highlighted in the water column, as we showed that 76% of the observed individuals have bioluminescence capability. More than 97% of Cnidarians were bioluminescent, and 9 of the 13 taxonomic categories were found to be bioluminescent dominant. The percentage of bioluminescent animals is remarkably uniform over depth. Moreover, the proportion of bioluminescent and non-bioluminescent animals within taxonomic groups changes with depth for Ctenophora, Scyphozoa, Chaetognatha, and Crustacea. Given these results, bioluminescence has to be considered an important ecological trait from the surface to the deep-sea.

  17. Quantification of bioluminescence from the surface to the deep sea demonstrates its predominance as an ecological trait

    PubMed Central

    Martini, Séverine; Haddock, Steven H. D.

    2017-01-01

    The capability of animals to emit light, called bioluminescence, is considered to be a major factor in ecological interactions. Because it occurs across diverse taxa, measurements of bioluminescence can be powerful to detect and quantify organisms in the ocean. In this study, 17 years of video observations were recorded by remotely operated vehicles during surveys off the California Coast, from the surface down to 3,900 m depth. More than 350,000 observations are classified for their bioluminescence capability based on literature descriptions. The organisms represented 553 phylogenetic concepts (species, genera or families, at the most precise taxonomic level defined from the images), distributed within 13 broader taxonomic categories. The importance of bioluminescent marine taxa is highlighted in the water column, as we showed that 76% of the observed individuals have bioluminescence capability. More than 97% of Cnidarians were bioluminescent, and 9 of the 13 taxonomic categories were found to be bioluminescent dominant. The percentage of bioluminescent animals is remarkably uniform over depth. Moreover, the proportion of bioluminescent and non-bioluminescent animals within taxonomic groups changes with depth for Ctenophora, Scyphozoa, Chaetognatha, and Crustacea. Given these results, bioluminescence has to be considered an important ecological trait from the surface to the deep-sea. PMID:28374789

  18. Modulation of endogenous β-tubulin isotype expression as a result of human βIII cDNA transfection into prostate carcinoma cells

    PubMed Central

    Ranganathan, S; McCauley, R A; Dexter, D W; Hudes, G R

    2001-01-01

    Increases of individual β tubulin isotypes in antimicrotubule drug resistant cell lines have been reported by several laboratories. We have previously described elevations in βIII and βIVa isotypes in estramustine and paclitaxel resistant human prostate carcinoma cells. To investigate further the function of β tubulin isotypes in antimicrotubule drug response, human prostate carcinoma cells that normally have very low to undetectable levels of βIII were stably transfected with βIII cDNA in pZeoSV system. An 18 bp haemagglutinin (HA) epitope tag was added at the 3′ end prior to cloning into the vector. Cells were transfected with pZeoSV or pZeoSV-βIII plasmids and selected in the presence of Zeocin. Immunofluorescent staining of the transfectant cells have shown significant expression and incorporation of HA-tagged βIII tubulin into cellular microtubules. Quantitation of Western blots revealed the HA-tagged βIII levels to be approximately 7-fold higher than the vector control cells. RT-PCR analysis confirmed the increase at the transcript level and also revealed a collateral increase of βII and βIVb transcripts. Cell viability assays indicated that sensitivity of βIII transfected cells to various antimicrotubule agents was similar to vector transfected cells: IC50 values for estramustine, paclitaxel, colchicine and vinblastine were 4 μM, 4 nM, 22 nM and 2 nM, respectively for both cell lines. Thus, overexpression of βIII isotype in human prostate carcinoma cells by stable transfection failed to confer antimicrotubule drug resistance to these cells. Counterregulatory increases of endogenous βII and βIVb tubulin isotypes in these βIII transfected cells may be a compensatory mechanism used by the cells to overcome the effects of elevated βIII levels on the cellular microtubules. These results highlight the difficulty in isolating the contribution of single tubulin isotypes in drug response studies. © 2001 Cancer Research Campaign http

  19. 106 ASSESSMENT OF ANTI-BACTERIAL EFFECTS OF PEGYLATED SILVER-COATED CARBON NANOTUBES ON CAUSATIVE BACTERIA OF BOVINE INFERTILITY USING BIOLUMINESCENCE IMAGING SYSTEM.

    PubMed

    Park, S; Chaudhari, A A; Pillai, S; Singh, S R; Willard, S T; Ryan, P L; Feugang, J M

    2016-01-01

    Pathogenic bacteria including Escherichia coli and Salmonella sp. are the major causative agents of endometritis and can cause infertility in livestock animals. Antibiotics are commonly used to terminate bacterial infections, but the development of bacterial antibiotic resistance is often encountered. Nanotechnology associated with silver nanoparticles has been highlighted as an alternative anti-bacterial agent, and pegylated silver-coated single-walled carbon nanotubes have high anti-bacterial effects and are non-toxic to human and murine cells in vitro. Here we verified whether a real-time bioluminescence monitoring system could be an alternative tool to assess anti-bacterial effects of nanotubes in a noninvasive approach. Escherichia coli and Salmonella sp. were transfected with plasmids containing constructs for luciferase enzyme (LuxCDABE) and substrate (luciferin) to create self-illuminating bioluminescent bacteria. Pathogens were grown in LB broth at 37°C, adjusted to 10(7) cfumL(-1), and placed in 96-well plates for treatments. Pegylated (pSWCNTs-Ag) and non-pegylated (SWCNTs-Ag) nanotubes were prepared and added to culture wells at various concentrations (31.25-125µgmL(-1)). The control group corresponded to bacteria without nanotubes (0µgmL(-1)). Anti-bacterial effects of nanotubes were determined every 10min until 1h, then every 30min up to 6h incubation through optical density (600nm) measurements and bioluminescence imaging (BLI) and quantification using an IVIS system. Optical density and BLI data were compared at each time-point using 2-way ANOVA, with P<0.05 set for significance. Bioluminescence signals emitted by both bacteria stains appeared within 10min of incubation. Thereafter, control bacteria showed exponential growth that was detected as early as 25min post-incubation. Bioluminescence imaging revealed dose-dependent anti-bacterial activities of both pSWCNTs-Ag and SWCNTs-Ag on each E. coli and Salmonella sp. (P<0.05). Contrary to BLI, the

  20. Quorum Sensing Influences Vibrio harveyi Growth Rates in a Manner Not Fully Accounted For by the Marker Effect of Bioluminescence

    PubMed Central

    Nackerdien, Zeena E.; Keynan, Alexander; Bassler, Bonnie L.; Lederberg, Joshua; Thaler, David S.

    2008-01-01

    Background The light-emitting Vibrios provide excellent material for studying the interaction of cellular communication with growth rate because bioluminescence is a convenient marker for quorum sensing. However, the use of bioluminescence as a marker is complicated because bioluminescence itself may affect growth rate, e.g. by diverting energy. Methodology/Principal Findings The marker effect was explored via growth rate studies in isogenic Vibrio harveyi (Vh) strains altered in quorum sensing on the one hand, and bioluminescence on the other. By hypothesis, growth rate is energy limited: mutants deficient in quorum sensing grow faster because wild type quorum sensing unleashes bioluminescence and bioluminescence diverts energy. Findings reported here confirm a role for bioluminescence in limiting Vh growth rate, at least under the conditions tested. However, the results argue that the bioluminescence is insufficient to explain the relationship of growth rate and quorum sensing in Vh. A Vh mutant null for all genes encoding the bioluminescence pathway grew faster than wild type but not as fast as null mutants in quorum sensing. Vh quorum sensing mutants showed altered growth rates that do not always rank with their relative increase or decrease in bioluminescence. In addition, the cell-free culture fluids of a rapidly growing Vibrio parahaemolyticus (Vp) strain increased the growth rate of wild type Vh without significantly altering Vh's bioluminescence. The same cell-free culture fluid increased the bioluminescence of Vh quorum mutants. Conclusions/Significance The effect of quorum sensing on Vh growth rate can be either positive or negative and includes both bioluminescence-dependent and independent components. Bioluminescence tends to slow growth rate but not enough to account for the effects of quorum sensing on growth rate. PMID:18301749

  1. Activation of Prn-p gene and stable transfection of Prn-p cDNA in leukemia MEL and neuroblastoma N2a cells increased production of PrP(C) but not prevented DNA fragmentation initiated by serum deprivation.

    PubMed

    Gougoumas, Dimitrios D; Vizirianakis, Ioannis S; Triviai, Ioanna N; Tsiftsoglou, Asterios S

    2007-05-01

    Prion protein (PrP(C)) via its isoform PrP(SC) is involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). We observed that murine erythroleukemia (MEL) cells arrested in phase G(1) undergo transcriptional activation of Prn-p gene. Here, we explored the potential role of activation of Prn-p gene and cytosolic accumulation of PrP(C) in growth arrest, differentiation, and apoptotic DNA fragmentation by stably transfecting MEL and N2a cells with Prn-p cDNA. Stably transfected MEL cells (clones # 6, 12, 20, 38, and 42) were assessed for growth and differentiation, while clones N2a13 and N2a8 of N2a cells for growth and apoptosis by flow cytometry using Annexin V and propidium iodide (PI). Our results indicate that (a) Induction of terminal differentiation of stably transfected MEL cells led to growth arrest, activation of Prn-p gene, concomitant expression of transfected Prn-p cDNA, suppression of bax gene, cytosolic accumulation of PrP(C), and DNA fragmentation. The latter was also induced in non-differentiated MEL cells growing under serum-free conditions; (b) similarly, serum deprivation promoted growth arrest, apoptosis/necrosis associated with DNA fragmentation in parental N2a and N2a13 cells that produced relative high level of PrP(C) and not PrP(SC). These data indicate that activation of Prn-p gene and expression of transfected Prn-p cDNA in cells of both hematopoietic and neuronal origin occurred concomitantly, and led to cytosolic accumulation of PrP(C) and DNA damage induced by serum deprivation. PrP(C) production failed to protect DNA fragmentation induced by serum deprivation. The question how does PrP(C) contribute to growth arrest and DNA fragmentation is discussed.

  2. Reporter cell activity within hydrogel constructs quantified from oxygen-independent bioluminescence.

    PubMed

    Lambrechts, Dennis; Roeffaers, Maarten; Kerckhofs, Greet; Hofkens, Johan; Van de Putte, Tom; Schrooten, Jan; Van Oosterwyck, Hans

    2014-09-01

    By providing a three-dimensional (3D) support to cells, hydrogels offer a more relevant in vivo tissue-like environment as compared to two-dimensional cell cultures. Hydrogels can be applied as screening platforms to investigate in 3D the role of biochemical and biophysical cues on cell behaviour using bioluminescent reporter cells. Gradients in oxygen concentration that result from the interplay between molecular transport and cell metabolism can however cause substantial variability in the observed bioluminescent reporter cell activity. To assess the influence of these oxygen gradients on the emitted bioluminescence for various hydrogel geometries, a combined experimental and modelling approach was implemented. We show that the applied model is able to predict oxygen gradient independent bioluminescent intensities which correlate better to the experimentally determined viable cell numbers, as compared to the experimentally measured bioluminescent intensities. By analysis of the bioluminescence reaction dynamics we obtained a quantitative description of cellular oxygen metabolism within the hydrogel, which was validated by direct measurements of oxygen concentration within the hydrogel. Bioluminescence peak intensities can therefore be used as a quantitative measurement of reporter cell activity within a hydrogel, but an unambiguous interpretation of these intensities requires a compensation for the influence of cell-induced oxygen gradients on the luciferase activity.

  3. Evaluation of an improved bioluminescence assay for the detection of bacteria in soy milk.

    PubMed

    Shinozaki, Yohei; Sato, Jun; Igarashi, Toshinori; Suzuki, Shigeya; Nishimoto, Kazunori; Harada, Yasuhiro

    2013-01-01

    Because soy milk is nutrient rich and nearly neutral in pH, it favors the growth of microbial contaminants. To ensure that soy milk meets food-safety standards, it must be pasteurized and have its sterility confirmed. ATP bioluminescence assay has become a widely accepted means of detecting food microorganisms. However, the high background bioluminescence intensity of soy milk has rendered it unsuitable for ATP analysis. Here, we tested the efficacy of an improved pre-treated bioluminescence assay on soy milk. By comparing background bioluminescence intensities obtained by the conventional and improved methods, we demonstrated that our method significantly reduces soy milk background bioluminescence. The dose-response curve of the assay was tested with serial dilutions of Bacillus sp. culture. An extremely strong log-linear relation between the bioluminescence intensity relative light units and colony formation units CFU/ml emerged for the tested strain. The detection limit of the assay was estimated as 5.2×10(3) CFU/ml from the dose-response curve and an imposed signal limit was three times the background level. The results showed that contaminated samples could be easily detected within 24 h using our improved bioluminescence assay.

  4. Bioluminescence in the ghost fungus Omphalotus nidiformis does not attract potential spore dispersing insects.

    PubMed

    Weinstein, Philip; Delean, Steven; Wood, Tom; Austin, Andrew D

    2016-12-01

    Bioluminescence has been known from fungi since ancient times, but little work has been done to establish its potential role. There is evidence that some bioluminescent fungi differentially attract potential spore-dispersing insects, and we aimed to establish if this was the case for the ghost fungus, Omphalotus nidiformis (Agaricales,Marasmiaceae), a widespread Australian temperate zone species. We examined three corroborative lines of evidence: circadian rhythmicity of bioluminescence; field-recorded insect abundance at the time of basidiome production; and attractiveness of glowing fungi to flying insects. Basidiomes glowed continuously day and night, and were present in winter (June-July) when insect abundance was low. To assess attractiveness, we deployed sticky-traps in open woodland in the absence of light pollution, in Treatment (baited with fresh bioluminescent O. nidiformis) and Control pairs, for 480 trap-hours on moonless nights. There was no statistical difference in mean insect abundance between Treatment and Control traps (mean 0.33 and 0.54 individuals per trap night, respectively). To interpret these results, we provide a brief review of competing hypotheses for fungal bioluminescence, and conclude that for some fungi, bioluminescence may be an incidental by-product of metabolism rather than conferring any selective advantage. It is possible that the role of bioluminescence differs among evolutionary lineages of fungi and/or with attributes of their growth environments that could affect spore dispersal, such as wind and insect abundance.

  5. Foraging in the darkness of the Southern Ocean: influence of bioluminescence on a deep diving predator.

    PubMed

    Vacquié-Garcia, Jade; Royer, François; Dragon, Anne-Cécile; Viviant, Morgane; Bailleul, Frédéric; Guinet, Christophe

    2012-01-01

    How non-echolocating deep diving marine predators locate their prey while foraging remains mostly unknown. Female southern elephant seals (SES) (Mirounga leonina) have vision adapted to low intensity light with a peak sensitivity at 485 nm. This matches the wavelength of bioluminescence produced by a large range of marine organisms including myctophid fish, SES's main prey. In this study, we investigated whether bioluminescence provides an accurate estimate of prey occurrence for SES. To do so, four SES were satellite-tracked during their post-breeding foraging trip and were equipped with Time-Depth-Recorders that also recorded light levels every two seconds. A total of 3386 dives were processed through a light-treatment model that detected light events higher than ambient level, i.e. bioluminescence events. The number of bioluminescence events was related to an index of foraging intensity for SES dives deep enough to avoid the influence of natural ambient light. The occurrence of bioluminescence was found to be negatively related to depth both at night and day. Foraging intensity was also positively related to bioluminescence both during day and night. This result suggests that bioluminescence likely provides SES with valuable indications of prey occurrence and might be a key element in predator-prey interactions in deep-dark marine environments.

  6. Near-infrared bioluminescent proteins for two-color multimodal imaging

    PubMed Central

    Rumyantsev, Konstantin A.; Turoverov, Konstantin K.; Verkhusha, Vladislav V.

    2016-01-01

    Bioluminescence imaging became a widely used technique for noninvasive study of biological processes in small animals. Bioluminescent probes with emission in near-infrared (NIR) spectral region confer the advantage of having deep tissue penetration capacity. However, there are a very limited number of currently available luciferases that exhibit NIR bioluminescence. Here, we engineered two novel chimeric probes based on RLuc8 luciferase fused with iRFP670 and iRFP720 NIR fluorescent proteins. Due to an intramolecular bioluminescence resonance energy transfer (BRET) between RLuc8 and iRFPs, the chimeric luciferases exhibit NIR bioluminescence with maxima at 670 nm and 720 nm, respectively. The 50 nm spectral shift between emissions of the two iRFP chimeras enables combined multicolor bioluminescence imaging (BLI) and the respective multicolor fluorescence imaging (FLI) of the iRFPs. We show that for subcutaneously implanted cells, NIR bioluminescence provided a 10-fold increase in sensitivity compared to NIR FLI. In deep tissues, NIR BLI enabled detection of as low as 104 cells. Both BLI and FLI allowed monitoring of tumor growth and metastasis from early to late stages. Multimodal imaging, which combines concurrent BLI and FLI, provides continuous spatiotemporal analysis of metastatic cells in animals, including their localization and quantification. PMID:27833162

  7. Effect of low-level alpha-radiation on bioluminescent assay systems of various complexity.

    PubMed

    Rozhko, Tatiana V; Kudryasheva, Nadezhda S; Kuznetsov, Alexander M; Vydryakova, Galina A; Bondareva, Lydia G; Bolsunovsky, Alexander Ya

    2007-01-01

    This study addresses the effects of low-level alpha-radiation on bioluminescent assay systems of different levels of organization: in vivo and in vitro. Three bioluminescent assay systems are used: intact bacteria, lyophilized bacteria, and bioluminescent system of coupled enzyme reactions. Solutions of 241Am(NO3)3 are used as a source of alpha-radiation. It has been shown that activation processes predominate in all the three bioluminescent assay systems subjected to short-term exposure (20-55 h) and inhibition processes in the systems subjected to longer-term exposure to radiation. It has been found that these effects are caused by the radiation component of 241Am3+ impact. The intensity of the 241Am3+ effect on the bioluminescent assay systems has been shown to depend on the 241Am3+ concentration, level of organization and integrity of the bioluminescent assay system. The bioluminescent assay systems in vivo have been found to be highly sensitive to 241Am3+ (up to 10(-17) M).

  8. Photodynamic inactivation of recombinant bioluminescent Escherichia coli by cationic porphyrins under artificial and solar irradiation.

    PubMed

    Alves, Eliana; Carvalho, Carla M B; Tomé, João P C; Faustino, Maria A F; Neves, Maria G P M S; Tomé, Augusto C; Cavaleiro, José A S; Cunha, Angela; Mendo, Sónia; Almeida, Adelaide

    2008-11-01

    A faster and simpler method to monitor the photoinactivation process of Escherichia coli involving the use of recombinant bioluminescent bacteria is described here. Escherichia coli cells were transformed with luxCDABE genes from the marine bioluminescent bacterium Vibrio fischeri and the recombinant bioluminescent indicator strain was used to assess, in real time, the effect of three cationic meso-substituted porphyrin derivatives on their metabolic activity, under artificial (40 W m(-2)) and solar irradiation (approximately 620 W m(-2)). The photoinactivation of bioluminescent E. coli is effective (>4 log bioluminescence decrease) with the three porphyrins used, the tricationic porphyrin Tri-Py+-Me-PF being the most efficient compound. The photoinactivation process is efficient both with solar and artificial light, for the three porphyrins tested. The results show that bioluminescence analysis is an efficient and sensitive approach being, in addition, more affordable, faster, cheaper and much less laborious than conventional methods. This approach can be used as a screening method for bacterial photoinactivation studies in vitro and also for the monitoring of the efficiency of novel photosensitizer molecules. As far as we know, this is the first study involving the use of bioluminescent bacteria to monitor the antibacterial activity of porphyrins under environmental conditions.

  9. Foraging in the Darkness of the Southern Ocean: Influence of Bioluminescence on a Deep Diving Predator

    PubMed Central

    Vacquié-Garcia, Jade; Royer, François; Dragon, Anne-Cécile; Viviant, Morgane; Bailleul, Frédéric; Guinet, Christophe

    2012-01-01

    How non-echolocating deep diving marine predators locate their prey while foraging remains mostly unknown. Female southern elephant seals (SES) (Mirounga leonina) have vision adapted to low intensity light with a peak sensitivity at 485 nm. This matches the wavelength of bioluminescence produced by a large range of marine organisms including myctophid fish, SES’s main prey. In this study, we investigated whether bioluminescence provides an accurate estimate of prey occurrence for SES. To do so, four SES were satellite-tracked during their post-breeding foraging trip and were equipped with Time-Depth-Recorders that also recorded light levels every two seconds. A total of 3386 dives were processed through a light-treatment model that detected light events higher than ambient level, i.e. bioluminescence events. The number of bioluminescence events was related to an index of foraging intensity for SES dives deep enough to avoid the influence of natural ambient light. The occurrence of bioluminescence was found to be negatively related to depth both at night and day. Foraging intensity was also positively related to bioluminescence both during day and night. This result suggests that bioluminescence likely provides SES with valuable indications of prey occurrence and might be a key element in predator-prey interactions in deep-dark marine environments. PMID:22952706

  10. Bioluminescence imaging: a shining future for cardiac regeneration

    PubMed Central

    Roura, Santiago; Gálvez-Montón, Carolina; Bayes-Genis, Antoni

    2013-01-01

    Advances in bioanalytical techniques have become crucial for both basic research and medical practice. One example, bioluminescence imaging (BLI), is based on the application of natural reactants with light-emitting capabilities (photoproteins and luciferases) isolated from a widespread group of organisms. The main challenges in cardiac regeneration remain unresolved, but a vast number of studies have harnessed BLI with the discovery of aequorin and green fluorescent proteins. First described in the luminous hydromedusan Aequorea victoria in the early 1960s, bioluminescent proteins have greatly contributed to the design and initiation of ongoing cell-based clinical trials on cardiovascular diseases. In conjunction with advances in reporter gene technology, BLI provides valuable information about the location and functional status of regenerative cells implanted into numerous animal models of disease. The purpose of this review was to present the great potential of BLI, among other existing imaging modalities, to refine effectiveness and underlying mechanisms of cardiac cell therapy. We recount the first discovery of natural primary compounds with light-emitting capabilities, and follow their applications to bioanalysis. We also illustrate insights and perspectives on BLI to illuminate current efforts in cardiac regeneration, where the future is bright. PMID:23402217

  11. Smartphone-based low light detection for bioluminescence application

    PubMed Central

    Kim, Huisung; Jung, Youngkee; Doh, Iyll-Joon; Lozano-Mahecha, Roxana Andrea; Applegate, Bruce; Bae, Euiwon

    2017-01-01

    We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an analyte. A simple cradle that houses the smartphone, sample tube, and collection lens supports the measuring platform, while noise reduction by ensemble averaging simultaneously lowers the background and enhances the signal from emitted photons. Five different types of smartphones, both Android and iOS devices, were tested, and the top two candidates were used to evaluate luminescence from the bioluminescent reporter Pseudomonas fluorescens M3A. The best results were achieved by OnePlus One (android), which was able to detect luminescence from ~106 CFU/mL of the bio-reporter, which corresponds to ~107 photons/s with 180 seconds of integration time. PMID:28067287

  12. Firefly bioluminescence: a mechanistic approach of luciferase catalyzed reactions.

    PubMed

    Marques, Simone M; Esteves da Silva, Joaquim C G

    2009-01-01

    Luciferase is a general term for enzymes catalyzing visible light emission by living organisms (bioluminescence). The studies carried out with Photinus pyralis (firefly) luciferase allowed the discovery of the reaction leading to light production. It can be regarded as a two-step process: the first corresponds to the reaction of luciferase's substrate, luciferin (LH(2)), with ATP-Mg(2+) generating inorganic pyrophosphate and an intermediate luciferyl-adenylate (LH(2)-AMP); the second is the oxidation and decarboxylation of LH(2)-AMP to oxyluciferin, the light emitter, producing CO(2), AMP, and photons of yellow-green light (550- 570 nm). In a dark reaction LH(2)-AMP is oxidized to dehydroluciferyl-adenylate (L-AMP). Luciferase also shows acyl-coenzyme A synthetase activity, which leads to the formation of dehydroluciferyl-coenzyme A (L-CoA), luciferyl-coenzyme A (LH(2)-CoA), and fatty acyl-CoAs. Moreover luciferase catalyzes the synthesis of dinucleoside polyphosphates from nucleosides with at least a 3'-phosphate chain plus an intact terminal pyrophosphate moiety. The LH(2) stereospecificity is a particular feature of the bioluminescent reaction where each isomer, D-LH(2) or L-LH(2), has a specific function. Practical applications of the luciferase system, either in its native form or with engineered proteins, encloses the analytical assay of metabolites like ATP and molecular biology studies with luc as a reporter gene, including the most recent and increasing field of bioimaging.

  13. Enhanced Landweber algorithm via Bregman iterations for bioluminescence tomography

    NASA Astrophysics Data System (ADS)

    Xia, Yi; Zhang, Meng

    2014-09-01

    Bioluminescence tomography (BLT) is an important optical molecular imaging modality aimed at visualizing physiological and pathological processes at cellular and molecular levels. While the forward process of light propagation is described by the diffusion approximation to radiative transfer equation, BLT is the inverse problem to reconstruct the 3D localization and quantification of internal bioluminescent sources distribution. Due to the inherent ill-posedness of the BLT problem, regularization is generally indispensable to obtain more favorable reconstruction. In particular, total variation (TV) regularization is known to be effective for piecewise-constant source distribution which can permit sharp discontinuities and preserve edges. However, total variation regularization generally suffers from the unsatisfactory staircasing effect. In this work, we introduce the Bregman iterative regularization to alleviate this degeneration and enhance the numerical reconstruction of BLT. Based on the existing Landweber method (LM), we put forward the Bregman-LM-TV algorithm for BLT. Numerical experiments are carried out and preliminary simulation results are reported to evaluate the proposed algorithms. It is found that Bregman-LM-TV can significantly outperform the individual Landweber method for BLT when the source distribution is piecewise-constant.

  14. Smartphone-based low light detection for bioluminescence application

    NASA Astrophysics Data System (ADS)

    Kim, Huisung; Jung, Youngkee; Doh, Iyll-Joon; Lozano-Mahecha, Roxana Andrea; Applegate, Bruce; Bae, Euiwon

    2017-01-01

    We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an analyte. A simple cradle that houses the smartphone, sample tube, and collection lens supports the measuring platform, while noise reduction by ensemble averaging simultaneously lowers the background and enhances the signal from emitted photons. Five different types of smartphones, both Android and iOS devices, were tested, and the top two candidates were used to evaluate luminescence from the bioluminescent reporter Pseudomonas fluorescens M3A. The best results were achieved by OnePlus One (android), which was able to detect luminescence from ~106 CFU/mL of the bio-reporter, which corresponds to ~107 photons/s with 180 seconds of integration time.

  15. Isolation and development of bioluminescent reporter phages for bacterial dysentery.

    PubMed

    Schofield, D A; Wray, D J; Molineux, I J

    2015-02-01

    Shigellosis is a significant cause of morbidity and mortality worldwide, most notably amongst children. Moreover, there is a global increase in the occurrence of multidrug-resistant isolates, including the epidemic and pandemic Shigella dysenteriae type 1 strain. We developed a bioluminescent reporter phage assay to facilitate detection and simultaneously determine antibiotic susceptibility. A Shigella flexneri phage (Shfl25875) was isolated from environmental wastewater and characterized by DNA sequencing. Shfl25875 is T4-like, harbors a 169,062-bp genome, and grows on most (28/29) S. flexneri strains and all 12 S. dysenteriae type 1 strains tested. The genes encoding bacterial luciferase were integrated into the Shfl25875 genome to create a "light-tagged" phage capable of transducing a bioluminescent phenotype to infected cells. Shfl25875::luxAB rapidly detects cultured isolates with high sensitivity. Specificity experiments indicate that the reporter does not respond to Shigella boydii, non-type 1 S. dysenteriae strains, and most non-Shigella Enterobacteriaceae. Shfl25875::luxAB generates ampicillin and ciprofloxacin susceptibility profiles that are similar to the standard Clinical and Laboratory Standards Institute (CLSI) growth microdilution method, but in a significantly shorter time. In addition, the reporter phage detects Shigella in mock-infected stool. This new reporter phage shows promise as a tool for the detection of cultured isolates or complex clinical samples.

  16. Smartphone-based low light detection for bioluminescence application.

    PubMed

    Kim, Huisung; Jung, Youngkee; Doh, Iyll-Joon; Lozano-Mahecha, Roxana Andrea; Applegate, Bruce; Bae, Euiwon

    2017-01-09

    We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an analyte. A simple cradle that houses the smartphone, sample tube, and collection lens supports the measuring platform, while noise reduction by ensemble averaging simultaneously lowers the background and enhances the signal from emitted photons. Five different types of smartphones, both Android and iOS devices, were tested, and the top two candidates were used to evaluate luminescence from the bioluminescent reporter Pseudomonas fluorescens M3A. The best results were achieved by OnePlus One (android), which was able to detect luminescence from ~10(6) CFU/mL of the bio-reporter, which corresponds to ~10(7) photons/s with 180 seconds of integration time.

  17. Metabolic imaging in tumours by means of bioluminescence.

    PubMed Central

    Tamulevicius, P.; Streffer, C.

    1995-01-01

    A bioluminescence technique involving single photon imaging was used to quantify the spatial distribution of the metabolites ATP, glucose and lactate in cryosections of various solid tumours and normal tissue. Each section was covered with an enzyme cocktail linking the metabolite in question to luciferase with light emission proportional to the metabolite concentration. The photons emitted are imaged directly through a microscope and an imaging photon counting system. In some cases, good agreement was observed between the distribution of relatively high concentrations of ATP and glucose in viable cell regions of the periphery, while the reverse was seen in more necrotic tumour centres with comparatively high lactate levels. In general, lactate was distributed more diffusely over the sections while ATP was more highly localised and glucose assumed an intermediate pattern. In contrast to the large degree of heterogeneity seen in tumours, distribution patterns of metabolites were much more homogeneous in normal tissue, such as heart muscle. Mean values for metabolite levels in cryosections using bioluminescence are in good agreement with those obtained from the same tumour by conventional methods. Images Figure 2 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7577454

  18. Aequorin fusion proteins as bioluminescent tracers for competitive immunoassays

    NASA Astrophysics Data System (ADS)

    Mirasoli, Mara; Michelini, Elisa; Deo, Sapna K.; Dikici, Emre; Roda, Aldo; Daunert, Sylvia

    2004-06-01

    The use of bio- and chemiluminescence for the development of quantitative binding assays offers undoubted advantages over other detection systems, such as spectrophotometry, fluorescence, or radioactivity. Indeed, bio- and chemiluminescence detection provides similar, or even better, sensitivity and detectability than radioisotopes, while avoiding the problems of health hazards, waste disposal, and instability associated with the use of radioisotopes. Among bioluminescent labels, the calcium-activated photoprotein aequorin, originally isolated from Aequorea victoria and today available as a recombinant product, is characterized by very high detectability, down to attomole levels. It has been used as a bioluminescent label for developing a variety of highly sensitive immunoassays, using various analyte-aequorin conjugation strategies. When the analyte is a protein or a peptide, genetic engineering techniques can be used to produce protein fusions where the analyte is in-frame fused with aequorin, thus producing homogeneous one-to-one conjugation products, available in virtually unlimited amount. Various assays were developed using this strategy: a short review of the most interesting applications is presented, as well as the cloning, purification and initial characterization of an endothelin-1-aequorin conjugate suitable for developing a competitive immunoassay for endothelin-1, a potent vasoconstrictor peptide, involved in hypertension.

  19. Bioluminescence imaging: a shining future for cardiac regeneration.

    PubMed

    Roura, Santiago; Gálvez-Montón, Carolina; Bayes-Genis, Antoni

    2013-06-01

    Advances in bioanalytical techniques have become crucial for both basic research and medical practice. One example, bioluminescence imaging (BLI), is based on the application of natural reactants with light-emitting capabilities (photoproteins and luciferases) isolated from a widespread group of organisms. The main challenges in cardiac regeneration remain unresolved, but a vast number of studies have harnessed BLI with the discovery of aequorin and green fluorescent proteins. First described in the luminous hydromedusan Aequorea victoria in the early 1960s, bioluminescent proteins have greatly contributed to the design and initiation of ongoing cell-based clinical trials on cardiovascular diseases. In conjunction with advances in reporter gene technology, BLI provides valuable information about the location and functional status of regenerative cells implanted into numerous animal models of disease. The purpose of this review was to present the great potential of BLI, among other existing imaging modalities, to refine effectiveness and underlying mechanisms of cardiac cell therapy. We recount the first discovery of natural primary compounds with light-emitting capabilities, and follow their applications to bioanalysis. We also illustrate insights and perspectives on BLI to illuminate current efforts in cardiac regeneration, where the future is bright.

  20. Bioluminescent properties of obelin and aequorin with novel coelenterazine analogues.

    PubMed

    Gealageas, Ronan; Malikova, Natalia P; Picaud, Sandrine; Borgdorff, Aren J; Burakova, Ludmila P; Brûlet, Philippe; Vysotski, Eugene S; Dodd, Robert H

    2014-04-01

    The main analytical use of Ca(2+)-regulated photoproteins from luminous coelenterates is for real-time non-invasive visualization of intracellular calcium concentration ([Ca(2+)]i) dynamics in cells and whole organisms. A limitation of this approach for in vivo deep tissue imaging is the fact that blue light emitted by the photoprotein is highly absorbed by tissue. Seven novel coelenterazine analogues were synthesized and their effects on the bioluminescent properties of recombinant obelin from Obelia longissima and aequorin from Aequorea victoria were evaluated. Only analogues having electron-donating groups (m-OCH3 and m-OH) on the C6 phenol moiety or an extended resonance system at the C8 position (1-naphthyl and α-styryl analogues) showed a significant red shift of light emission. Of these, only the α-styryl analogue displayed a sufficiently high light intensity to allow eventual tissue penetration. The possible suitability of this compound for in vivo assays was corroborated by studies with aequorin which allowed the monitoring of [Ca(2+)]i dynamics in cultured CHO cells and in hippocampal brain slices. Thus, the α-styryl coelenterazine analogue might be potentially useful for non-invasive, in vivo bioluminescence imaging in deep tissues of small animals.

  1. [ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum].

    PubMed

    Alekserova, L É; Alenina, K A; Efremenko, E N; Mazhul', M M; Piskunova, N F; Ismailov, A D

    2014-01-01

    Bioluminescence activity and ATP pool were investigated in the culture of psychrophilic bacteria Photobacterium phosphoreum collected-from the exponential and stationary growth phases, as well as immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost a constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilizedl cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h): Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation by the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.

  2. Bioluminescence regenerative cycle (BRC) system for nucleic acid quantification assays

    NASA Astrophysics Data System (ADS)

    Hassibi, Arjang; Lee, Thomas H.; Davis, Ronald W.; Pourmand, Nader

    2003-07-01

    A new label-free methodology for nucleic acid quantification has been developed where the number of pyrophosphate molecules (PPi) released during polymerization of the target nucleic acid is counted and correlated to DNA copy number. The technique uses the enzymatic complex of ATP-sulfurylase and firefly luciferase to generate photons from PPi. An enzymatic unity gain positive feedback is also implemented to regenerate the photon generation process and compensate any decay in light intensity by self regulation. Due to this positive feedback, the total number of photons generated by the bioluminescence regenerative cycle (BRC) can potentially be orders of magnitude higher than typical chemiluminescent processes. A system level kinetic model that incorporates the effects of contaminations and detector noise was used to show that the photon generation process is in fact steady and also proportional to the nucleic acid quantity. Here we show that BRC is capable of detecting quantities of DNA as low as 1 amol (10-18 mole) in 40μlit aqueous solutions, and this enzymatic assay has a controllable dynamic range of 5 orders of magnitude. The sensitivity of this technology, due to the excess number of photons generated by the regenerative cycle, is not constrained by detector performance, but rather by possible PPi or ATP (adenosine triphosphate) contamination, or background bioluminescence of the enzymatic complex.

  3. Targeted DNA degradation using a CRISPR device stably carried in the host genome

    PubMed Central

    Caliando, Brian J.; Voigt, Christopher A.

    2015-01-01

    Once an engineered organism completes its task, it is useful to degrade the associated DNA to reduce environmental release and protect intellectual property. Here we present a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-defined DNA. This enables engineered regions to be obscured when the cell enters a new environment. DNAi is based on type-IE CRISPR biochemistry and a synthetic CRISPR array defines the DNA target(s). When the input is on, plasmid DNA is degraded 108-fold. When the genome is targeted, this causes cell death, reducing viable cells by a factor of 108. Further, the CRISPR nuclease can direct degradation to specific genomic regions (for example, engineered or inserted DNA), which could be used to complicate recovery and sequencing efforts. DNAi can be stably carried in an engineered organism, with no impact on cell growth, plasmid stability or DNAi inducibility even after passaging for >2 months. PMID:25988366

  4. Single-column Model Intercomparison for a Stably Stratified Atmospheric Boundary Layer

    NASA Technical Reports Server (NTRS)

    Cuxart, J.; Holtslag, A. A. M.; Steeneveld, G-J; Beare, R. J.; Bazile, E.; Beljaars, A.; Cheng, A.; Conangla, L.; Ek, M.; Freedman, F.; Hamdi, R.

    2004-01-01

    The parameterization of the stably stratified atmospheric boundary layer is a difficult issue, which has a large impact on the medium-range weather forecasts and on climate integrations. A non-strongly stratified arctic case is simulated by nineteen single-column turbulence schemes. The statistics from the Large-eddy simulation (LES) intercomparison made for the same case by eight different models are used as a guiding reference. The single-column parameterizations include research schemes and operational schemes from major forecast and climate research centres. First order schemes, a large number of turbulence kinetic energy closures, and other proposals have submitted results. There is a large spread in the results; in general, the operational schemes mix more efficiently than the research ones, and the TKE and other higher order closures give results closer to the LES statistics. The sensitivities of the schemes to the parameters of their turbulence closures are partially explored.

  5. Targeted DNA degradation using a CRISPR device stably carried in the host genome.

    PubMed

    Caliando, Brian J; Voigt, Christopher A

    2015-05-19

    Once an engineered organism completes its task, it is useful to degrade the associated DNA to reduce environmental release and protect intellectual property. Here we present a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-defined DNA. This enables engineered regions to be obscured when the cell enters a new environment. DNAi is based on type-IE CRISPR biochemistry and a synthetic CRISPR array defines the DNA target(s). When the input is on, plasmid DNA is degraded 10(8)-fold. When the genome is targeted, this causes cell death, reducing viable cells by a factor of 10(8). Further, the CRISPR nuclease can direct degradation to specific genomic regions (for example, engineered or inserted DNA), which could be used to complicate recovery and sequencing efforts. DNAi can be stably carried in an engineered organism, with no impact on cell growth, plasmid stability or DNAi inducibility even after passaging for >2 months.

  6. Direct numerical simulation of intermittent turbulence in stably stratified plane Couette flow

    NASA Astrophysics Data System (ADS)

    Mortikov, Evgeny

    2016-11-01

    This work uses direct numerical simulation approach to investigate intermittent turbulence in stably stratified plane Couette flow for Reynolds numbers, based on the channel height and relative wall speed between top and bottom walls, up to 105. Results show that the transition to intermittent turbulence under strong stratification is associated with the formation of secondary counter-rotating roll-like structures elongated in the spanwise direction and organized in two rows corresponding to lower and upper walls of the channel. The ordering of rolls define spatially confined alternating regions of laminar and turbulent flow. The spanwise length of this vortices increases with the increase of the bulk Richardson number and defines an additional constraint on the computational box size. This study describes direct numerical simulation results in spanwise-extended computational domains, where the turbulent intermittent regime is sustained without relaminarization for sufficiently higher bulk Richardson numbers than previously reported.

  7. Continuous wave terahertz spectroscopy system with stably tunable beat source using optical switch

    NASA Astrophysics Data System (ADS)

    Eom, Joo Beom; Kim, Chihoon; Ahn, Jaesung

    2017-01-01

    A tunable beat source has been made using an optical switch module. A stably-tunable beat source for continuous wave terahertz spectroscopy system was implemented by simply connecting 16 coaxial distributed feedback laser diodes to an optical switch. The terahertz frequency was rapidly changed without frequency drifts by changing the optical path. The continuous wave terahertz frequency was tuned from 0.05 to 0.8 THz in steps of 50 GHz or 0.4 nm. We measured continuous wave terahertz waveforms emitted from the photomixers using the switched optical beat source. We also calculated the terahertz frequency peaks by taking fast Fourier transforms of the measured terahertz waveforms. By equipping the implemented tunable beat source with an optical switch, a continuous wave terahertz spectroscopy system was constructed and used to demonstrate the feasibility of continuous wave terahertz spectroscopy for nondestructive tests using the spectra of two type of Si wafers with different resistivity.

  8. Dispersion in an open-cut coal mine in stably stratified flow

    NASA Astrophysics Data System (ADS)

    Grainger, Clive; Meroney, Robert N.

    1993-02-01

    Discharges from combustion within a coal pit which occur during night-time inversion conditions may result in stagnant accumulation of smoke and dangerous gases which could inhibit mining operations. A wind-tunnel model study was performed to identify the range of flow and mixing conditions which could exist when stably stratified atmospheric surface flows pass over a large open pit. Flow penetration into the pit depended upon approach-flow stability (Froude number) and the strength of the thermal inversion within the coal pit. Measurements of wind speed and temperature were made upwind, within and downwind of the pit. Concentration measurements were made within the pit, of surface sources released along pit walls. Pollutant levels were found to be strong functions of the approach-flow pit Froude number, source location, and release time.

  9. Long-range sediment transport in the world's oceans by stably stratified turbidity currents

    NASA Astrophysics Data System (ADS)

    Kneller, Benjamin; Nasr-Azadani, Mohamad M.; Radhakrishnan, Senthil; Meiburg, Eckart

    2016-12-01

    Submarine fans, supplied primarily by turbidity currents, constitute the largest sediment accumulations on Earth. Generally accepted models of turbidity current behavior imply they should dissipate rapidly on the very small gradients of submarine fans, thus their persistence over long distances is enigmatic. We present numerical evidence, constrained by published field data, suggesting that turbidity currents traveling on low slopes and carrying fine particles have a stably stratified shear layer along their upper interface, which dramatically reduces dissipation and entrainment of ambient fluid, allowing the current to propagate over long distances. We propose gradient Richardson number as a useful criterion to discriminate between the different behaviors exhibited by turbidity currents on high and low slopes.

  10. A synthetic luciferin improves in vivo bioluminescence imaging of gene expression in cardiovascular brain regions.

    PubMed

    Simonyan, Hayk; Hurr, Chansol; Young, Colin N

    2016-10-01

    Bioluminescence imaging is an effective tool for in vivo investigation of molecular processes. We have demonstrated the applicability of bioluminescence imaging to spatiotemporally monitor gene expression in cardioregulatory brain nuclei during the development of cardiovascular disease, via incorporation of firefly luciferase into living animals, combined with exogenous d-luciferin substrate administration. Nevertheless, d-luciferin uptake into the brain tissue is low, which decreases the sensitivity of bioluminescence detection, particularly when considering small changes in gene expression in tiny central areas. Here, we tested the hypothesis that a synthetic luciferin, cyclic alkylaminoluciferin (CycLuc1), would be superior to d-luciferin for in vivo bioluminescence imaging in cardiovascular brain regions. Male C57B1/6 mice underwent targeted delivery of an adenovirus encoding the luciferase gene downstream of the CMV promoter to the subfornical organ (SFO) or paraventricular nucleus of hypothalamus (PVN), two crucial cardioregulatory neural regions. While bioluminescent signals could be obtained following d-luciferin injection (150 mg/kg), CycLuc1 administration resulted in a three- to fourfold greater bioluminescent emission from the SFO and PVN, at 10- to 20-fold lower substrate concentrations (7.5-15 mg/kg). This CycLuc1-mediated enhancement in bioluminescent emission was evident early following substrate administration (i.e., 6-10 min) and persisted for up to 1 h. When the exposure time was reduced from 60 s to 1,500 ms, minimal signal in the PVN was detectable with d-luciferin, whereas bioluminescent images could be reliably captured with CycLuc1. These findings demonstrate that bioluminescent imaging with the synthetic luciferin CycLuc1 provides an improved physiological genomics tool to investigate molecular events in discrete cardioregulatory brain nuclei.

  11. Evaluation of parameterization for turbulent fluxes of momentum and heat in stably stratified surface layers

    NASA Astrophysics Data System (ADS)

    Sodemann, H.; Foken, Th.

    2003-04-01

    General Circulation Models calculate the energy exchange between surface and atmosphere by means of parameterisations for turbulent fluxes of momentum and heat in the surface layer. However, currently implemented parameterisations after Louis (1979) create large discrepancies between predictions and observational data, especially in stably stratified surface layers. This work evaluates a new surface layer parameterisation proposed by Zilitinkevich et al. (2002), which was specifically developed to improve energy flux predictions in stable stratification. The evaluation comprises a detailed study of important surface layer characteristics, a sensitivity study of the parameterisation, and a direct comparison to observational data from Antarctica and predictions by the Louis (1979) parameterisation. The stability structure of the stable surface layer was found to be very complex, and strongly influenced fluxes in the surface layer. The sensitivity study revealed that the new parameterisation depends strongly on the ratio between roughness length and roughness temperature, which were both observed to be very variable parameters. The comparison between predictions and measurements showed good agreement for momentum fluxes, but large discrepancies for heat fluxes. A stability dependent evaluation of selected data showed better agreement for the new parameterisation of Zilitinkevich et al. (2002) than for the Louis (1979) scheme. Nevertheless, this comparison underlines the need for more detailed and physically sound concepts for parameterisations of heat fluxes in stably stratified surface layers. Zilitinkevich, S. S., V. Perov and J. C. King (2002). "Near-surface turbulent fluxes in stable stratification: Calculation techniques for use in General Circulation Models." Q. J. R. Meteorol. Soc. 128(583): 1571--1587. Louis, J. F. (1979). "A Parametric Model of Vertical Eddy Fluxes in the Atmosphere." Bound.-Layer Meteor. 17(2): 187--202.

  12. Structure-activity relationship in cationic lipid mediated gene transfection.

    PubMed

    Niculescu-Duvaz, Dan; Heyes, James; Springer, Caroline J

    2003-07-01

    Non-viral synthetic vectors for gene delivery represent a safer alternative to viral vectors. Their main drawback is the low transfection efficiency, especially in vivo. Among the non-viral vectors currently in use, the cationic liposomes composed of cationic lipids are the most common. This review discusses the physicochemical properties of cationic lipids, the formation, macrostructure and specific parameters of the corresponding formulated liposomes, and the effect of all these parameters on transfection efficiency. The optimisation of liposomal vectors requires both the understanding of the biological variables involved in the transfection process, and the effect of the structural elements of the cationic lipids on these biological variables. The biological barriers relevant for in vitro and in vivo transfection are identified, and solutions to overcome them based on rational design of the cationic lipids are discussed. The review focuses on the relationship between the structure of the cationic lipid and the transfection activity. The structure is analysed in a modular manner. The hydrophobic domain, the cationic head group, the backbone that acts as a scaffold for the other domains, the linkers between backbone, hydrophobic domain and cationic head group, the polyethyleneglycol chains and the targeting moiety are identified as distinct elements of the cationic lipids used in gene therapy. The main chemical functionalities used to built these domains, as well as overall molecular features such as architecture and geometry, are presented. Studies of structure-activity relationships of each cationic lipid domain, including the authors', and the trends identified by these studies, help furthering the understanding of the mechanism governing the formation and behaviour of cationic liposomes in gene delivery, and therefore the rational design of new improved cationic lipids vectors capable of achieving clinical significance.

  13. DEVELOPMENT OF A DUAL MODALITY TOMOGRAPHIC IMAGING SYSTEM FOR BIOLUMINESCENCE AND PET

    SciTech Connect

    CHATZIIOANNOU, ARION

    2011-12-21

    The goal of this proposal was to develop a new hybrid imaging modality capable to simultaneously image optical bioluminescence signals, as well as radionuclide emissions from the annihilation of positrons originating from molecular imaging probes in preclinical mouse models. This new technology enables the simultaneous in-vivo measurements of both emissions that could be produced from a single or a combination of two different biomarkers. It also facilitates establishing the physical limitations of bioluminescence imaging, its tomographic and spectral image reconstruction potential and the quantification of bioluminescence signals.

  14. Ultraweak bioluminescence dynamics and singlet oxygen correlations during injury repair in sweet potato

    NASA Astrophysics Data System (ADS)

    Hossu, Marius; Ma, Lun; Chen, Wei

    2011-03-01

    Ultraweak bioluminescence at the level of hundreds of photons per second per square centimeter after cutting injury of sweet potato was investigated. A small emission peak immediate after cutting and a later and higher peak were observed. Selective singlet oxygen inhibitors and sensors have been use to study the contribution of singlet oxygen during the curing process, demonstrating increased presence of singlet oxygen during and after the late bioemission peak. It was confirmed that singlet oxygen has direct contribution to ultraweak bioluminescence but also induces the formation of other exited luminescent species that are responsible for the recorded bioluminescence.

  15. A novel reconstruction algorithm for bioluminescent tomography based on Bayesian compressive sensing

    NASA Astrophysics Data System (ADS)

    Wang, Yaqi; Feng, Jinchao; Jia, Kebin; Sun, Zhonghua; Wei, Huijun

    2016-03-01

    Bioluminescence tomography (BLT) is becoming a promising tool because it can resolve the biodistribution of bioluminescent reporters associated with cellular and subcellular function through several millimeters with to centimeters of tissues in vivo. However, BLT reconstruction is an ill-posed problem. By incorporating sparse a priori information about bioluminescent source, enhanced image quality is obtained for sparsity based reconstruction algorithm. Therefore, sparsity based BLT reconstruction algorithm has a great potential. Here, we proposed a novel reconstruction method based on Bayesian compressive sensing and investigated its feasibility and effectiveness with a heterogeneous phantom. The results demonstrate the potential and merits of the proposed algorithm.

  16. High resolution in vitro bioluminescence imaging using a multimodal optical system

    NASA Astrophysics Data System (ADS)

    Altabella, L.; Gigliotti, C. R.; Perani, L.; Crippa, M. P.; Boschi, F.; Spinelli, A. E.

    2016-01-01

    Bioluminescence in vitro studies are usually performed with dedicated microscopes. In this work, we developed a novel image recovery algorithm and a multimodal system prototype to perform bioluminescence microscopy. We performed a feasibility study using GEANT4 Monte Carlo (MC) simulation of bioluminescent cells acquired at low SNR frames and processed using a Super Resolution Regularization Algorithm (SRRA). The method was also tested using in vitro cell acquisition. The results obtained with MC simulations showed an improvement in the spatial resolution from 90 μ m to 10 μ m and from 110 μ m to 13 μ m for in vitro imaging of mesothelioma cells.

  17. Unanimous Model for Describing the Fast Bioluminescence Kinetics of Ca(2+) -regulated Photoproteins of Different Organisms.

    PubMed

    Eremeeva, Elena V; Bartsev, Sergey I; van Berkel, Willem J H; Vysotski, Eugene S

    2017-03-01

    Upon binding their metal ion cofactors, Ca(2+) -regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca(2+) -regulated photoproteins-aequorin from Aequorea victoria, clytin from Clytia gregaria, mitrocomin from Mitrocoma cellularia and obelins from Obelia longissima and Obelia geniculata-demonstrate the same bioluminescent kinetics pattern. Based on these findings, for the first time we propose a unanimous kinetic model describing the bioluminescence mechanism of Ca(2+) -regulated photoproteins.

  18. Study of firefly luciferin oxidation and isomerism as possible inhibition pathways for firefly bioluminescence

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

    Firefly bioluminescence presents a light emitting profile with a form of a flash, due to the firefly luciferase-catalyzed formation of inhibitory products. These impair the binding of the substrate luciferin to the active site of the enzyme. However, this luciferase catalyzed pathways may not be the only ones responsible for the flash profile. The oxidation and isomerisation of the substrate luciferin lead to the formation of compounds that are also known inhibitors of firefly bioluminescence. So, the objective of this Letter was to analyze if these reactions could be capable of interfering with the bioluminescence reaction.

  19. Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

    PubMed Central

    Brown, Laura E.; Fuchs, Celine; Nicholson, Martin W.; Stephenson, F. Anne; Thomson, Alex M.; Jovanovic, Jasmina N.

    2014-01-01

    Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed

  20. QM/MM study on the light emitters of aequorin chemiluminescence, bioluminescence, and fluorescence: a general understanding of the bioluminescence of several marine organisms.

    PubMed

    Chen, Shu-Feng; Ferré, Nicolas; Liu, Ya-Jun

    2013-06-24

    Aequorea victoria is a type of jellyfish that is known by its famous protein, green fluorescent protein (GFP), which has been widely used as a probe in many fields. Aequorea has another important protein, aequorin, which is one of the members of the EF-hand calcium-binding protein family. Aequorin has been used for intracellular calcium measurements for three decades, but its bioluminescence mechanism remains largely unknown. One of the important reasons is the lack of clear and reliable knowledge about the light emitters, which are complex. Several neutral and anionic forms exist in chemiexcited, bioluminescent, and fluorescent states and are connected with the H-bond network of the binding cavity in the protein. We first theoretically investigated aequorin chemiluminescence, bioluminescence, and fluorescence in real proteins by performing hybrid quantum mechanics and molecular mechanics methods combined with a molecular dynamics method. For the first time, this study reported the origin and clear differences in the chemiluminescence, bioluminescence and fluorescence of aequorin, which is important for understanding the bioluminescence not only of jellyfish, but also of many other marine organisms (that have the same coelenterazine caved in different coelenterazine-type luciferases).

  1. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    SciTech Connect

    Gruenhagen, Jason Alan

    2003-01-01

    The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca2+ imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca2+ signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K+ and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol

  2. cAMP-activated chloride channels in a CFTR-transfected pancreatic adenocarcinoma-derived cell line, pANS6.

    PubMed

    Smith, A N; Wardle, C J; Winpenny, J P; Verdon, B; Gray, M A; Argent, B E; Harris, A

    1995-06-09

    Pancreatic adenocarcinoma cell lines rarely express the CFTR gene, despite the high levels of CFTR protein that are present in primary pancreatic duct cells. We have attempted to generate a non-CF pancreatic adenocarcinoma cell line that stably produces high levels of CFTR mRNA and protein by transfecting a vector containing the CFTR cDNA, driven by a strong mammalian promoter, into the poorly differentiated pancreatic adenocarcinoma cell line, Panc-1. The pANS6 pancreatic duct cell line expresses substantial levels of CFTR mRNA, but little CFTR protein. Despite this we were able to detect low conductance chloride channels in 40% of patches, stimulated with cAMP, that have similar biophysical properties to CFTR.

  3. Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells

    PubMed Central

    González, Cecilia; Esteban, Rosa; Canals, Carme; Muñiz-Díaz, Eduardo; Nogués, Núria

    2016-01-01

    Background The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs), which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories. Methods We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua). High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment. Results TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies. Conclusions Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs. PMID:27603310

  4. The Theoretical Estimation of the Bioluminescent Efficiency of the Firefly via a Nonadiabatic Molecular Dynamics Simulation.

    PubMed

    Yue, Ling; Lan, Zhenggang; Liu, Ya-Jun

    2015-02-05

    The firefly is famous for its high bioluminescent efficiency, which has attracted both scientific and public attention. The chemical origin of firefly bioluminescence is the thermolysis of the firefly dioxetanone anion (FDO(-)). Although considerable theoretical research has been conducted, and several mechanisms were proposed to elucidate the high efficiency of the chemi- and bioluminescence of FDO(-), there is a lack of direct experimental and theoretical evidence. For the first time, we performed a nonadiabatic molecular dynamics simulation on the chemiluminescent decomposition of FDO(-) under the framework of the trajectory surface hopping (TSH) method and theoretically estimated the chemiluminescent quantum yield. The TSH simulation reproduced the gradually reversible charge-transfer initiated luminescence mechanism proposed in our previous study. More importantly, the current study, for the first time, predicted the bioluminescence efficiency of the firefly from a theoretical viewpoint, and the theoretical prediction efficiency is in good agreement with experimental measurements.

  5. Facile synthesis of gold-silver alloy nanoparticles for application in metal enhanced bioluminescence.

    PubMed

    Abhijith, K S; Sharma, Richa; Ranjan, Rajeev; Thakur, M S

    2014-07-01

    In the present study we explored metal enhanced bioluminescence in luciferase enzymes for the first time. For this purpose a simple and reproducible one pot synthesis of gold-silver alloy nanoparticles was developed. By changing the molar ratio of tri-sodium citrate and silver nitrate we could synthesize spherical Au-Ag colloids of sizes ranging from 10 to 50 nm with a wide range of localized surface plasmon resonance (LSPR) peaks (450-550 nm). The optical tunability of the Au-Ag colloids enabled their effective use in enhancement of bioluminescence in a luminescent bacterium Photobacterium leiognathi and in luciferase enzyme systems from fireflies and bacteria. Enhancement of bioluminescence was 250% for bacterial cells, 95% for bacterial luciferase and 52% for firefly luciferase enzyme. The enhancement may be a result of energy transfer or plasmon induced enhancement. Such an increase can lead to higher sensitivity in detection of bioluminescent signals with potential applications in bio-analysis.

  6. Interaction between in vivo bioluminescence and extracellular electron transfer in Shewanella woodyi via charge and discharge.

    PubMed

    Tian, Xiaochun; Zhao, Feng; You, Lexing; Wu, Xuee; Zheng, Zhiyong; Wu, Ranran; Jiang, Yanxia; Sun, Shigang

    2017-01-18

    Extracellular electron transfer (EET) and bioluminescence are both important for microbial growth and metabolism, but the mechanism of interaction between EET and bioluminescence is poorly understood. Herein, we demonstrate an exclusively respiratory luminous bacterium, Shewanella woodyi, which possesses EET ability and electron communication at the interface of S. woodyi and solid substrates via charge and discharge methods. Using an electro-chemiluminescence apparatus, our results confirmed that the FMN/FMNH2 content and the redox status of cytochrome c conjointly regulated the bioluminescence intensity when the potential of an indium-tin oxide electrode was changed. More importantly, this work revealed that there is an interaction between the redox reaction of single cells and bioluminescence of group communication via the EET pathway.

  7. Bioluminescence: A Potentially Convergent Signature of Life in Future Exploration of Europa's Subsurface Ocean

    NASA Astrophysics Data System (ADS)

    Flores Martinez, C. L.

    2014-02-01

    This presentation deals with theoretical and evolutionary aspects pertaining to the nature and degree of biological complexity that is expectable among putative organisms on Europa. Bioluminescence is suggested as a new type of biosignature.

  8. Bioluminescent luciferase-modified magnetic nanoparticles as potential imaging agents for mammalian spermatozoa detection and tracking

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Nanoparticles have emerged as key materials for developing applications in nanomedicine, nanobiotechnology, bioimaging and theranostics. Existing bioimaging technologies include bioluminescent resonance energy transfer-conjugated quantum dots (BRET-QDs). Despite the current use of BRET-Q...

  9. Bioluminescent reporter bacterium for toxicity monitoring in biological wastewater treatment systems

    SciTech Connect

    Kelly, C.J.; Lajoie, C.A.; Layton, A.C.; Sayler, G.S.

    1999-01-01

    Toxic shock due to certain chemical loads in biological wastewater treatment systems can result in death of microorganisms and loss of floc structure. To overcome the limitations of existing approaches to toxicity monitoring, genes encoding enzymes for light production were inserted to a bacterium (Shk 1) isolated from activated sludge. The Shk 1 bioreporter indicated a toxic response to concentrations of cadmium, 2,4-dinitrophenol, and hydroquinone by reductions in initial levels of bioluminescence on exposure to the toxicant. The decrease in bioluminescence was more severe with increasing toxicant concentration. Bioluminescence did not decrease in response to ethanol concentrations up to 1,000 mg/L or to pH conditions between 6.1 and 7.9. A continuous toxicity monitoring system using this bioreporter was developed for influent wastewater and tested with hydroquinone. The reporter exhibited a rapid and proportional decrease in bioluminescence in response to increasing hydroquinone concentrations.

  10. Bacterial bioluminescence response to long-term exposure to reverse osmosis treated effluents from dye industries.

    PubMed

    Ravindran, J; Manikandan, B; Shirodkar, P V; Francis, K X; Mani Murali, R; Vethamony, P

    2014-10-01

    The bacterial bioluminescence assay is one of the novel means for toxicity detection. The bioluminescence response of 2 marine bioluminescent bacteria was tested upon their long-term exposure to 9 different reverse osmosis (RO) rejects with varying chemical composition sampled from various dye industries. Bioluminescent bacteria were cultured in the RO reject samples, at different concentrations, and their growth rate and luminescence was measured for 24 h. The RO reject samples caused sublethal effects upon exposure and retarded the growth of bacteria, confirming their toxic nature. Further, continuation of the exposure showed that the initial luminescence, though reduced, recovered and increased beyond the control cultures irrespective of cell density, and finally decreased once again. The present study emphasizes the need of evolving a long-term exposure assay and shows that the method followed in this study is suitable to evaluate the toxicants that exert delayed toxicity, using lower concentrations of toxicants as well as coloured samples.

  11. Determination of the Genetic Diversity of Different Bioluminescent Bacteria by Pulsed-Field Gel Electrophoresis (PFGE)

    PubMed Central

    Ersoy Omeroglu, Esra

    2015-01-01

    Background: There are 4 different genera (i.e. Vibrio, Aliivibrio, Photobacterium, and Shewanella) in the new classification of bioluminescent bacteria. The mechanism of bioluminescence has yet to be fully elucidated. Therefore, the determination of physiological and genetic characteristics of bioluminescent bacteria isolated from different sources is very important. Pulsed-Field Gel Electrophoresis (PFGE) has the highest discriminatory power among the different molecular typing methods for the investigation of the clonal relationships between bacteria. For the PFGE analysis of bioluminescent bacteria, the NotI-HF™ is the method of choice among the restriction enzymes. Objectives: The present study aimed to determine genetic relatedness via PFGE in 41 bioluminescent bacteria (belonging to 10 different species) isolated and identified from various marine sources. Materials and Methods: Different bioluminescent bacteria (i.e. Vibrio gigantis, V. azureus, V. harveyi, V. lentus, V. crassostreae, V. orientalis, Aliivibrio logei, A. fischeri, Shewanella woodyi, and Photobacterium kishitanii) were analyzed by PFGE using the NotI-HF™ restriction enzyme. The whole DNA of the strains embedded into the agarose plugs was digested with enzyme at 37°C for 30 minutes. CHEF-Mapper PFGE system was used for electrophoresis and band profile of the strains for the NotI-HF™ restriction enzyme were analyzed by Bio-Profil-1D++ software (Vilber Lourmat) at 10% homology coefficient. Results: Although all experiments were performed three times, four of forty-one bioluminescent strains (V. gigantis E-16, H-16 and S3W46 strains and A. fischeri E-4 strain) could not be typed by PFGE technique with NotI-HF™ enzyme. While only two strains (V. crassostreae H-12 and H-19 strains) were exhibiting same band pattern profiles (100% genome homology), thirty-six different PFGE band patterns were obtained. Pattern homologies changed between 66% - 92%, 73% - 83% and 49% - 100% for V. gigantis, V

  12. Transfection of a human glioblastoma cell line with liver-type glutaminase (LGA) down-regulates the expression of DNA-repair gene MGMT and sensitizes the cells to alkylating agents.

    PubMed

    Szeliga, Monika; Zgrzywa, Agata; Obara-Michlewska, Marta; Albrecht, Jan

    2012-11-01

    O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA-repair protein promoting resistance of tumor cells to alkylating chemotherapeutic agents. Glioma cells are particularly resistant to this class of drugs which include temozolomide (TMZ) and carmustine (BCNU). A previous study using the RNA microarray technique showed that decrease of MGMT mRNA stands out among the alterations in gene expression caused by the cell growth-depressing transfection of a T98G glioma cell line with liver-type glutaminase (LGA) [Szeliga et al. (2009) Glia, 57, 1014]. Here, we show that stably LGA-transfected cells (TLGA) exhibit decreased MGMT protein expression and activity as compared with non-transfected or mock transfected cells (controls). However, the decrease of expression occurs in the absence of changes in the methylation of the promoter region, indicating that LGA circumvents, by an as yet unknown route, the most common mechanism of MGMT silencing. TLGA turned out to be significantly more sensitive to treatment with 100-1000 μM of TMZ and BCNU in the acute cell growth inhibition assay (MTT). In the clonogenic survival assay, TLGA cells displayed increased sensitivity even to 10 μM TMZ and BCNU. Our results indicate that enrichment with LGA, in addition to inhibiting glioma growth, may facilitate chemotherapeutic intervention.

  13. Self-protection and self-similarity of the stably-stratified geophysical turbulence

    NASA Astrophysics Data System (ADS)

    Zilitinkevich, Sergej; Kleeorin, Nathan; Rogachevskii, Igor

    2014-05-01

    Following Richardson (1920), the effect of stratification on the shear-generated geophysical turbulence is determined by the gradient Richardson number Ri = (N/S)2, where Nis the Brunt-Vaisala frequency, S = dU/dz is vertical shear of the mean wind/current velocity U, and z is vertical coordinate. The concept of Richardson-number similarity postulates that dimensionless characteristics of turbulence are universal functions of Ri. Monin and Obukhov (1954) have proposed for the atmospheric surface layer a widely recognised Monin-Obukhov similarity theory (MOST). This theory postulates that dimensionless characteristics of turbulence are fully determined by the ratio z/L, where L = -u*3/Fb is the Obukhov length scale, u* is friction velocity and Fb is vertical turbulent flux of buoyancy. Nieuwstadt (1984) has employed local,z-dependent values of Fb and u* instead of the surface values, and demonstrated applicability of such version of MOST to the almost entire stably stratified planetary boundary layer. MOST is consistent with the Ri-similarity: in the surface layer Ri is a monotonously increasing function of z/L and vice versa (e.g., Sorbjan, 2010). In the strongly unstable stratification, MOST and Ri-similarity fail because of the self-organisation of convective turbulence (Elperin et al., 2006; Zilitinkevich et al., 2006). In this paper we employ the EFB turbulence closure theory (Zilitinkevich et al, 2013) together with available experimental, LES and DNS data to explain the most puzzling feature of the stably stratified geophysical turbulence, namely, its self-protection in very stable stratification, due to the counter-gradient heat-transfer mechanism missed in the traditional theory. We also explain the self-similarity of turbulence, due to the Kolmogorov's nature of dissipation for the turbulent kinetic energy (TKE), turbulent potential energy (TPE) and turbulent fluxes of heat and momentum. In non-steady regimes, traditional similarity criteria, such as z

  14. Photoporation and cell transfection using a violet diode laser

    NASA Astrophysics Data System (ADS)

    Paterson, L.; Agate, B.; Comrie, M.; Ferguson, R.; Lake, T. K.; Morris, J. E.; Carruthers, A. E.; Brown, C. T. A.; Sibbett, W.; Bryant, P. E.; Gunn-Moore, F.; Riches, A. C.; Dholakia, Kishan

    2005-01-01

    The introduction and subsequent expression of foreign DNA inside living mammalian cells (transfection) is achieved by photoporation with a violet diode laser. We direct a compact 405 nm laser diode source into an inverted optical microscope configuration and expose cells to 0.3 mW for 40 ms. The localized optical power density of ~1200 MW/m2 is six orders of magnitude lower than that used in femtosecond photoporation (~104 TW/m2). The beam perforates the cell plasma membrane to allow uptake of plasmid DNA containing an antibiotic resistant gene as well as the green fluorescent protein (GFP) gene. Successfully transfected cells then expand into clonal groups which are used to create stable cell lines. The use of the violet diode laser offers a new and simple poration technique compatible with standard microscopes and is the simplest method of laser-assisted cell poration reported to date.

  15. Roles of biogenic amines in regulating bioluminescence in the Australian glowworm Arachnocampa flava.

    PubMed

    Rigby, Lisa M; Merritt, David J

    2011-10-01

    The glowworm Arachnocampa flava is a carnivorous fly larva (Diptera) that uses light to attract prey into its web. The light organ is derived from cells of the Malpighian tubules, representing a bioluminescence system that is unique to the genus. Bioluminescence is modulated through the night although light levels change quite slowly compared with the flashing of the better-known fireflies (Coleoptera). The existing model for the neural regulation of bioluminescence in Arachnocampa, based on use of anaesthetics and ligations, is that bioluminescence is actively repressed during the non-glowing phase and the repression is partially released during the bioluminescence phase. The effect of the anaesthetic, carbon dioxide, on the isolated light organ from the present study indicates that the repression is at least partially mediated at the light organ itself rather than less directly through the central nervous system. Blocking of neural signals from the central nervous system through ligation leads to uncontrolled release of bioluminescence but light is emitted at relatively low levels compared with under anaesthesia. Candidate biogenic amines were introduced by several methods: feeding prey items injected with test solution, injecting the whole larva, injecting a ligated section containing the light organ or bathing the isolated light organ in test solution. Using these methods, dopamine, serotonin and tyramine do not affect bioluminescence output. Exposure to elevated levels of octopamine via feeding, injection or bathing of the isolated light organ indicates that it is involved in the regulation of repression. Administration of the octopamine antagonists phentolamine or mianserin results in very high bioluminescence output levels, similar to the effect of anaesthetics, but only mianserin acts directly on the light organ.

  16. New bioreactor for in situ simultaneous measurement of bioluminescence and cell density

    NASA Astrophysics Data System (ADS)

    Picart, Pascal; Bendriaa, Loubna; Daniel, Philippe; Horry, Habib; Durand, Marie-José; Jouvanneau, Laurent; Thouand, Gérald

    2004-03-01

    This article presents a new device devoted to the simultaneous measurement of bioluminescence and optical density of a bioluminescent bacterial culture. It features an optoelectronic bioreactor with a fully autoclavable module, in which the bioluminescent bacteria are cultivated, a modulated laser diode dedicated to optical density measurement, and a detection head for the acquisition of both bioluminescence and optical density signals. Light is detected through a bifurcated fiber bundle. This setup allows the simultaneous estimation of the bioluminescence and the cell density of the culture medium without any sampling. The bioluminescence is measured through a highly sensitive photomultiplier unit which has been photometrically calibrated to allow light flux measurements. This was achieved by considering the bioluminescence spectrum and the full optical transmission of the device. The instrument makes it possible to measure a very weak light flux of only a few pW. The optical density is determined through the laser diode and a photodiode using numerical synchronous detection which is based on the power spectrum density of the recorded signal. The detection was calibrated to measure optical density up to 2.5. The device was validated using the Vibrio fischeri bacterium which was cultivated under continuous culture conditions. A very good correlation between manual and automatic measurements processed with this instrument has been demonstrated. Furthermore, the optoelectronic bioreactor enables determination of the luminance of the bioluminescent bacteria which is estimated to be 6×10-5 W sr-1 m-2 for optical density=0.3. Experimental results are presented and discussed.

  17. Seasonal Changes of Bioluminescence in Photosynthetic and Heterotrophic Dinoflagellates at San Clemente Island

    DTIC Science & Technology

    2012-02-01

    the northern Sargasso Sea : seasonal and vertical distribution. Mar. Biol. 104: 153- 164 Seasonal Changes of Bioluminescence in Photosynthetic and...bioluminescence in the northern Sargasso Sea . Mar. Biol. 113: 329-339 Bityukov, E.P., Rybasov, V.P., Shaida, V.G. (1967). Annual variations of the...geographically. Dinoflagellates are most abundant in coastal waters and inland seas and are less abundant in the open ocean (Colebrook and Robinson, 1965

  18. Rapid antimicrobial susceptibility determination of uropathogens in clinical urine specimens by use of ATP bioluminescence.

    PubMed

    Ivancic, Vesna; Mastali, Mitra; Percy, Neil; Gornbein, Jeffrey; Babbitt, Jane T; Li, Yang; Landaw, Elliot M; Bruckner, David A; Churchill, Bernard M; Haake, David A

    2008-04-01

    We describe the first direct testing of the antimicrobial susceptibilities of bacterial pathogens in human clinical fluid samples by the use of ATP bioluminescence. We developed an ATP bioluminescence assay that eliminates somatic sources of ATP to selectively quantify the bacterial load in clinical urine specimens with a sensitivity of <1,000 CFU per milliliter. There was a log-log relationship between light emission and the numbers of CFU in clinical urine specimens. A clinical study was performed to evaluate the accuracy of the ATP bioluminescence assay for determination of the antimicrobial susceptibilities of uropathogens in clinical urine specimens tested in a blinded manner. ATP bioluminescent bacterial density quantitation was used to determine the inoculation volume in growth medium with and without antibiotics. After incubation at 37 degrees C for 120 min, the ATP bioluminescence assay was repeated to evaluate the uropathogen response to antibiotics. The ability of the ATP bioluminescence assay to discriminate between antimicrobial susceptibility and resistance was determined by comparison of the results obtained by the ATP bioluminescence assay with the results obtained by standard clinical microbiology methods. Receiver operator characteristic curves were used to determine the optimal threshold for discriminating between susceptibility and resistance. Susceptibility and resistance were correctly predicted in 87% and 95% of cases, respectively, for an overall unweighted accuracy of 91%, when the results were stratified by antibiotic. For samples in which the pathogen was susceptible, the accuracy improved to 95% when the results for samples with less than a 25-fold increase in the amount of bacterial ATP in the medium without antibiotics were excluded. These data indicate that a rapid bioluminescent antimicrobial susceptibility assay may be useful for the management of urinary tract infections.

  19. Luminol-based bioluminescence imaging of mouse mammary tumors.

    PubMed

    Alshetaiwi, Hamad S; Balivada, Sivasai; Shrestha, Tej B; Pyle, Marla; Basel, Matthew T; Bossmann, Stefan H; Troyer, Deryl L

    2013-10-05

    Polymorphonuclear neutrophils (PMNs) are the most abundant circulating blood leukocytes. They are part of the innate immune system and provide a first line of defense by migrating toward areas of inflammation in response to chemical signals released from the site. Some solid tumors, such as breast cancer, also cause recruitment and activation of PMNs and release of myeloperoxidase. In this study, we demonstrate that administration of luminol to mice that have been transplanted with 4T1 mammary tumor cells permits the detection of myeloperoxidase activity, and consequently, the location of the tumor. Luminol allowed detection of activated PMNs only two days after cancer cell transplantation, even though tumors were not yet palpable. In conclusion, luminol-bioluminescence imaging (BLI) can provide a pathway towards detection of solid tumors at an early stage in preclinical tumor models.

  20. Morphogenesis and bioluminescence in germination of red bean

    NASA Astrophysics Data System (ADS)

    Kai, Shoichi; Mitani, Tomohiko; Fujikawa, Masahiro

    1994-10-01

    Spontaneous bioluminescence and morphogenesis were investigated for the germination and the growth processes of a red bean seed under suppression of photosynthesis. Three types of shape in seed growth were observed in well controlled conditions: (1) no root hair and leaves, (2) with root hairs and leaves and (3) no root growth. In this article, growth dynamics for the first case was investigated. The average growth dynamics of the root length of a red bean after germination and its variance were well described by a simple logistic equation with a noise term. It was observed that the scaling property for the growth dynamics has held. Strong luminescence was observed at two inflection points of the logistic curve of the root growth. By the use of a two dimensional photon counting method, it was clarified that the strong emission was mainly radiated from the cell division zone near a root cap and rather less emission from an elongation area.

  1. Real-Time Bioluminescent Tracking of Cellular Population Dynamics

    SciTech Connect

    Close, Dan; Sayler, Gary Steven; Xu, Tingting; Ripp, Steven Anthony

    2014-01-01

    Cellular population dynamics are routinely monitored across many diverse fields for a variety of purposes. In general, these dynamics are assayed either through the direct counting of cellular aliquots followed by extrapolation to the total population size, or through the monitoring of signal intensity from any number of externally stimulated reporter proteins. While both viable methods, here we describe a novel technique that allows for the automated, non-destructive tracking of cellular population dynamics in real-time. This method, which relies on the detection of a continuous bioluminescent signal produced through expression of the bacterial luciferase gene cassette, provides a low cost, low time-intensive means for generating additional data compared to alternative methods.

  2. Enhanced Beetle Luciferase for High-Resolution Bioluminescence Imaging

    PubMed Central

    Nakajima, Yoshihiro; Yamazaki, Tomomi; Nishii, Shigeaki; Noguchi, Takako; Hoshino, Hideto; Niwa, Kazuki; Viviani, Vadim R.; Ohmiya, Yoshihiro

    2010-01-01

    We developed an enhanced green-emitting luciferase (ELuc) to be used as a bioluminescence imaging (BLI) probe. ELuc exhibits a light signal in mammalian cells that is over 10-fold stronger than that of the firefly luciferase (FLuc), which is the most widely used luciferase reporter gene. We showed that ELuc produces a strong light signal in primary cells and tissues and that it enables the visualization of gene expression with high temporal resolution at the single-cell level. Moreover, we successfully imaged the nucleocytoplasmic shuttling of importin α by fusing ELuc at the intracellular level. These results demonstrate that the use of ELuc allows a BLI spatiotemporal resolution far greater than that provided by FLuc. PMID:20368807

  3. Two-phase flow cell for chemiluminescence and bioluminescence measurements

    SciTech Connect

    Mullin, J.L.; Seitz, W.R.

    1984-01-01

    A new approach to two-phase CL (chemiluminescence) measurements is reported. A magnetically stirred reagent phase is separated from the analyte phase by a dialysis membrane so that only smaller molecules can go from one phase to the other. The system is designed so that the analyte phase flows through a spiral groove on an aluminum block that is flush against the dialysis membrane. As solution flows through the spiral grove, analyte diffuses into the reagent phase where it reacts to produce light. A simple model is developed to predict how this system will behave. Experimentally, the system is evaluated by using the luminol reaction catalyzed by peroxidase, the firefly reaction, and the bacterial bioluminescence reaction. 10 references, 4 tables, 6 figures.

  4. Real-Time Bioluminescent Tracking of Cellular Population Dynamics

    PubMed Central

    Close, Dan; Xu, Tingling; Ripp, Steven; Sayler, Gary

    2015-01-01

    Cellular population dynamics are routinely monitored across many diverse fields for a variety of purposes. In general, these dynamics are assayed either through the direct counting of cellular aliquots followed by extrapolation to the total population size, or through the monitoring of signal intensity from any number of externally stimulated reporter proteins. While both viable methods, here we describe a novel technique that allows for the automated, non-destructive tracking of cellular population dynamics in real-time. This method, which relies on the detection of a continuous bioluminescent signal produced through expression of the bacterial luciferase gene cassette, provides a low cost, low time-intensive means for generating additional data compared to alternative methods. PMID:24166372

  5. Preservative efficacy screening of pharmaceutical formulations using ATP bioluminescence.

    PubMed

    Kramer, Mateja; Suklje-Debeljak, Helena; Kmetec, Vojko

    2008-05-01

    The preservative challenge test is a method used to determine the efficacy of a preservation system in a pharmaceutical or cosmetic formulation. However, such testing is a labor-intensive, repetitive task often requiring days before results can be generated. Several alternatives to traditional colony-count techniques have been developed. A study using pure suspensions of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Candida albicans, and Aspergillus niger showed that the accuracy, repeatability, and linearity of the Pallchek luminometer ATP bioluminescence (ATP-B) system was equivalent to the traditional colony-count method. In any case, the method proved sensitive enough to follow the effect of preservatives on a number of test microorganisms, indicating the applicability of the ATP-B method for preservative screening studies in various pharmaceutical formulations.

  6. Bioluminescence microscopy: application to ATP measurements in single living cells

    NASA Astrophysics Data System (ADS)

    Brau, Frederic; Helle, Pierre; Bernengo, Jean C.

    1997-12-01

    Bioluminescence microscopy can be used to measure intracellular cofactors and ionic concentrations (Ca2+, K+, ATP, NADH), as an alternative to micro- spectrophotometry and micro-fluorimetry, due to the development of sensitive detectors (cooled photomultipliers tubes and CCD). The main limitation comes from the very small and brief intensity of the emitted light. Our instrumentation based on an inverted microscope, equipped with high aperture immersion lenses is presented. Light intensity measurements are carried out through a photomultiplier sorted for low dark current and cooled at -5 degree(s)C to reduce thermal noise. Our first aim is to quantify ATP on single living cells using the firefly luciferin-luciferase couple. Experimental and kinetic aspects are presented to emphasize the potentialities of the technique.

  7. Enhanced transfection of brain tumor suppressor genes by photochemical internalization

    NASA Astrophysics Data System (ADS)

    Chou, Chih H.; Sun, Chung-Ho; Zhou, Yi-Hong; Madsen, Steen J.; Hirschberg, Henry

    2011-03-01

    One of many limitations for cancer gene therapy is the inability of the therapeutic gene to transfect a sufficient number of tumor cells. Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. The utility of PCI for the delivery of a tumor suppressor gene (PAX-6) was investigated in monolayers and spheroids consisting of F98 rat glioma cells.

  8. Nanothermite-Based Microsystem for Drug Delivery and Cell Transfection

    DTIC Science & Technology

    2008-12-01

    presented. 2. EXPERIMENTAL 2.1 Nanothermite Preparation Nanothermite mixtures consisting of Bi2O3 nanoparticles and Al nanoparticles were used...for the transfection experiments reported herein. The Bi2O3 Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for the...particles were physically mixed in isopropanol (IPA) using ultrasonic agitation. Batches were mixed by dispersing 200mg of Bi2O3 in 1.5 mL of IPA by

  9. Whole-cell bioluminescent bioreporter sensing of foodborne toxicants

    NASA Astrophysics Data System (ADS)

    Ripp, Steve A.; Applegate, Bruce M.; Simpson, Michael L.; Sayler, Gary S.

    2001-03-01

    The presence of biologically derived toxins in foods is of utmost significance to food safety and human health concerns. Biologically active amines, referred to as biogenic amines, serve as a noteworthy example, having been implicated as the causative agent in numerous food poisoning episodes. Of the various biogenic amines encountered, histamine, putrescine, cadaverine, tyramine, tryptamine, beta-phenylethylamine, spermine, and spermidine are considered to be the most significant, and can be used as hygienic-quality indicators of food. Biogenic amines can be monitored using whole-cell bioluminescent bioreporters, which represent a family of genetically engineered microorganisms that generate visible light in response to specific chemical or physical agents in their environment. The light response occurs due to transcriptional activation of a genetically incorporated lux cassette, and can be measured using standard photomultiplier devices. We have successfully engineered a lux-based bioreporter capable of detecting and monitoring the biogenic amine beta-phenylethylamine. This research represents a biologically-based sensor technology that can be readily integrated into Hazard Analysis Critical Control Point programs to provide a rugged monitoring regime that can be uniformly applied for field-based and in-house laboratory quality control analyses. Since the bioreporter and biosensing elements are completely self-contained within the sensor design, this system provides ease of use, with operational capabilities realized by simply combining the food sample with the bioreporter and allowing the sensor to process the ensuing bioluminescent signal and communicate the results. The application of this technology to the critically important issue of food safety and hygienic quality represents a novel method for detecting, monitoring, and preventing biologically active toxins in food commodities.

  10. Iterative reconstruction for bioluminescence tomography with total variation regularization

    NASA Astrophysics Data System (ADS)

    Jin, Wenma; He, Yonghong

    2012-12-01

    Bioluminescence tomography(BLT) is an instrumental molecular imaging modality designed for the 3D location and quantification of bioluminescent sources distribution in vivo. In our context, the diffusion approximation(DA) to radiative transfer equation(RTE) is utilized to model the forward process of light propagation. Mathematically, the solution uniqueness does not hold for DA-based BLT which is an inverse source problem of partial differential equations and hence is highly ill-posed. In the current work, we concentrate on a general regularization framework for BLT with Bregman distance as data fidelity and total variation(TV) as regularization. Two specializations of the Bregman distance, the least squares(LS) distance and Kullback-Leibler(KL) divergence, which correspond to the Gaussian and Poisson environments respectively, are demonstrated and the resulting regularization problems are denoted as LS+TV and KL+TV. Based on the constrained Landweber(CL) scheme and expectation maximization(EM) algorithm for BLT, iterative algorithms for the LS+TV and KL+TV problems in the context of BLT are developed, which are denoted as CL-TV and EM-TV respectively. They are both essentially gradient-based algorithms alternatingly performing the standard CL or EM iteration step and the TV correction step which requires the solution of a weighted ROF model. Chambolle's duality-based approach is adapted and extended to solving the weighted ROF subproblem. Numerical experiments for a 3D heterogeneous mouse phantom are carried out and preliminary results are reported to verify and evaluate the proposed algorithms. It is found that for piecewise-constant sources both CL-TV and EM-TV outperform the conventional CL and EM algorithms for BLT.

  11. Regulation of Bioluminescence in Photobacterium leiognathi Strain KNH6

    PubMed Central

    Rader, Bethany A.; Stabb, Eric V.; Mandel, Mark J.

    2015-01-01

    ABSTRACT Bacterial bioluminescence is taxonomically restricted to certain proteobacteria, many of which belong to the Vibrionaceae. In the most well-studied cases, pheromone signaling plays a key role in regulation of light production. However, previous reports have indicated that certain Photobacterium strains do not use this regulatory method for controlling luminescence. In this study, we combined genome sequencing with genetic approaches to characterize the regulation of luminescence in Photobacterium leiognathi strain KNH6, an extremely bright isolate. Using transposon mutagenesis and screening for decreased luminescence, we identified insertions in genes encoding components necessary for the luciferase reaction (lux, lum, and rib operons) as well as in nine other loci. These additional loci encode gene products predicted to be involved in the tricarboxylic acid (TCA) cycle, DNA and RNA metabolism, transcriptional regulation, and the synthesis of cytochrome c, peptidoglycan, and fatty acids. The mutagenesis screen did not identify any mutants with disruptions of predicted pheromone-related loci. Using targeted gene insertional disruptions, we demonstrate that under the growth conditions tested, luminescence levels do not appear to be controlled through canonical pheromone signaling systems in this strain. IMPORTANCE Despite the long-standing interest in luminous bacteria, outside a few model organisms, little is known about the regulation and function of luminescence. Light-producing marine bacteria are widely distributed and have diverse lifestyles, suggesting that the control and significance of luminescence may be similarly diverse. In this study, we apply genetic tools to the study of regulation of light production in the extremely bright isolate Photobacterium leiognathi KNH6. Our results suggest an unusual lack of canonical pheromone-mediated control of luminescence and contribute to a better understanding of alternative strategies for regulation of a

  12. Application of nanostructured biochips for efficient cell transfection microarrays

    NASA Astrophysics Data System (ADS)

    Akkamsetty, Yamini; Hook, Andrew L.; Thissen, Helmut; Hayes, Jason P.; Voelcker, Nicolas H.

    2007-01-01

    Microarrays, high-throughput devices for genomic analysis, can be further improved by developing materials that are able to manipulate the interfacial behaviour of biomolecules. This is achieved both spatially and temporally by smart materials possessing both switchable and patterned surface properties. A system had been developed to spatially manipulate both DNA and cell growth based upon the surface modification of highly doped silicon by plasma polymerisation and polyethylene grafting followed by masked laser ablation for formation of a pattered surface with both bioactive and non-fouling regions. This platform has been successfully applied to transfected cell microarray applications with the parallel expression of genes by utilising its ability to direct and limit both DNA and cell attachment to specific sites. One of the greatest advantages of this system is its application to reverse transfection, whereupon by utilising the switchable adsorption and desorption of DNA using a voltage bias, the efficiency of cell transfection can be enhanced. However, it was shown that application of a voltage also reduces the viability of neuroblastoma cells grown on a plasma polymer surface, but not human embryonic kidney cells. This suggests that the application of a voltage may not only result in the desorption of bound DNA but may also affect attached cells. The characterisation of a DNA microarray by contact printing has also been investigated.

  13. Graphene and carbon nanotube nanocomposite for gene transfection.

    PubMed

    Hollanda, L M; Lobo, A O; Lancellotti, M; Berni, E; Corat, E J; Zanin, H

    2014-06-01

    Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5m(2)/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency.

  14. Towards gene therapy based on femtosecond optical transfection

    NASA Astrophysics Data System (ADS)

    Antkowiak, M.; Torres-Mapa, M. L.; McGinty, J.; Chahine, M.; Bugeon, L.; Rose, A.; Finn, A.; Moleirinho, S.; Okuse, K.; Dallman, M.; French, P.; Harding, S. E.; Reynolds, P.; Gunn-Moore, F.; Dholakia, K.

    2012-06-01

    Gene therapy poses a great promise in treatment and prevention of a variety of diseases. However, crucial to studying and the development of this therapeutic approach is a reliable and efficient technique of gene and drug delivery into primary cell types. These cells, freshly derived from an organ or tissue, mimic more closely the in vivo state and present more physiologically relevant information compared to cultured cell lines. However, primary cells are known to be difficult to transfect and are typically transfected using viral methods, which are not only questionable in the context of an in vivo application but rely on time consuming vector construction and may also result in cell de-differentiation and loss of functionality. At the same time, well established non-viral methods do not guarantee satisfactory efficiency and viability. Recently, optical laser mediated poration of cell membrane has received interest as a viable gene and drug delivery technique. It has been shown to deliver a variety of biomolecules and genes into cultured mammalian cells; however, its applicability to primary cells remains to be proven. We demonstrate how optical transfection can be an enabling technique in research areas, such as neuropathic pain, neurodegenerative diseases, heart failure and immune or inflammatory-related diseases. Several primary cell types are used in this study, namely cardiomyocytes, dendritic cells, and neurons. We present our recent progress in optimizing this technique's efficiency and post-treatment cell viability for these types of cells and discuss future directions towards in vivo applications.

  15. Use of bioluminescent Escherichia coli O157:H7 to study intra-protozoan survival of bacteria within Tetrahymena pyriformis.

    PubMed

    Nelson, Shona M; Cooper, Alison A A; Taylor, Elaine L; Salisbury, Vyvyan C

    2003-06-06

    A method was developed that enabled real-time monitoring of the uptake and survival of bioluminescent Escherichia coli O157 within the freshwater ciliate Tetrahymena pyriformis. Constitutively bioluminescent E. coli O157 pLITE27 was cocultured with T. pyriformis in nutrient-deficient (Chalkley's) and in nutrient-rich (proteose peptone, yeast extract) media. Non-internalised bacteria were inactivated by addition of colistin, indicated by a decline in bioluminescence. Protozoa were subsequently lysed with Triton X-100 which lead to a further drop in bioluminescence, consistent with release of live internal bacteria from T. pyriformis into the colistin-containing environment. Bioluminescence measurements for non-lysed cultures indicated that internalised E. coli O157 pLITE27 cells were only slowly digested by T. pyriformis, in both media, over the time period studied. The results suggest that bioluminescent bacteria are useful tools in the study of bacterial intra-protozoan survival.

  16. A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging

    PubMed Central

    Kuchimaru, Takahiro; Iwano, Satoshi; Kiyama, Masahiro; Mitsumata, Shun; Kadonosono, Tetsuya; Niwa, Haruki; Maki, Shojiro; Kizaka-Kondoh, Shinae

    2016-01-01

    In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models. PMID:27297211

  17. Discovery of a glowing millipede in California and the gradual evolution of bioluminescence in Diplopoda

    PubMed Central

    Marek, Paul E.; Moore, Wendy

    2015-01-01

    The rediscovery of the Californian millipede Xystocheir bistipita surprisingly reveals that the species is bioluminescent. Using molecular phylogenetics, we show that X. bistipita is the evolutionary sister group of Motyxia, the only genus of New World bioluminescent millipedes. We demonstrate that bioluminescence originated in the group’s most recent common ancestor and evolved by gradual, directional change through diversification. Because bioluminescence in Motyxia has been experimentally demonstrated to be aposematic, forewarning of the animal’s cyanide-based toxins, these results are contrary to aposematic theory and empirical evidence that a warning pattern cannot evolve gradually in unpalatable prey. However, gradual evolution of a warning pattern is plausible if faint light emission served another function and was co-opted as an aposematic signal later in the diversification of the genus. Luminescence in Motyxia stem-group taxa may have initially evolved to cope with reactive oxygen stress triggered by a hot, dry environment and was repurposed for aposematism by high-elevation crown-group taxa colonizing new habitats with varying levels of predation. The discovery of bioluminescence in X. bistipita and its pivotal phylogenetic location provides insight into the independent and repeated evolution of bioluminescence across the tree of life. PMID:25941389

  18. Second bioluminescence-activating component in the luminous fungus Mycena chlorophos.

    PubMed

    Teranishi, Katsunori

    2017-03-01

    Mycena chlorophos is an oxygen-dependent bioluminescent fungus. The mechanisms underlying its light emission are unknown. A component that increased the bioluminescence intensity of the immature living gills of M. chlorophos was isolated from mature M. chlorophos gills and chemically characterized. The bioluminescence-activating component was found to be trans-3,4-dihydroxycinnamic acid and its bioluminescence activation was highly structure-specific. (13) C- and (18) O-labelling studies using the immature living gills showed that trans-3,4-dihydroxycinnamic acid was synthesized from trans-4-hydroxycinnamic acid in the gills by hydroxylation with molecular oxygen as well as by the general metabolism, and trans-3,4-dihydroxycinnamic acid did not produce hispidin (detection-limit concentration: 10 pmol/1 g wet gill). Addition of 0.01 mM hispidin to the immature living gills generated no bioluminescence activation. These results suggested that the prompt bioluminescence activation resulting from addition of trans-3,4-dihydroxycinnamic acid could not be attributed to the generation of hispidin. Copyright © 2016 John Wiley & Sons, Ltd.

  19. A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging.

    PubMed

    Kuchimaru, Takahiro; Iwano, Satoshi; Kiyama, Masahiro; Mitsumata, Shun; Kadonosono, Tetsuya; Niwa, Haruki; Maki, Shojiro; Kizaka-Kondoh, Shinae

    2016-06-14

    In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models.

  20. ATP bioluminescence rapid detection of total viable count in soy sauce.

    PubMed

    Yan, Shou-Lei; Miao, Su-Na; Deng, Shao-Ya; Zou, Min-Juan; Zhong, Fo-Sheng; Huang, Wen-Biao; Pan, Si-Yi; Wang, Qing-Zhang

    2012-01-01

    The adenosine triphosphate (ATP) bioluminescence rapid determination method may be useful for enumerating the total viable count (TVC) in soy sauce, as it has been previously used in food and beverages for sanitation with good precision. However, many factors interfere with the correlation between total aerobic plate counts and ATP bioluminescence. This study investigated these interfering factors, including ingredients of soy sauce and bacteria at different physiological stages. Using the ATP bioluminescence method, TVC was obtained within 4 h, compared to 48 h required for the conventional aerobic plate count (APC) method. Our results also indicated a high correlation coefficient (r = 0.90) between total aerobic plate counts and ATP bioluminescence after filtration and resuscitation with special medium. The limit of quantification of the novel detection method is 100 CFU/mL; there is a good linear correlation between the bioluminescence intensity and TVC in soy sauce in the range 1 × 10(2) -3 × 10(4) CFU/mL and even wider. The method employed a luminescence recorder (Tristar LB-941) and 96-well plates and could analyse 50-100 samples simultaneously at low cost. In this study, we evaluated and eliminated the interfering factors and made the ATP bioluminescence rapid method available for enumerating TVC in soy sauce.

  1. Discovery of a glowing millipede in California and the gradual evolution of bioluminescence in Diplopoda.

    PubMed

    Marek, Paul E; Moore, Wendy

    2015-05-19

    The rediscovery of the Californian millipede Xystocheir bistipita surprisingly reveals that the species is bioluminescent. Using molecular phylogenetics, we show that X. bistipita is the evolutionary sister group of Motyxia, the only genus of New World bioluminescent millipedes. We demonstrate that bioluminescence originated in the group's most recent common ancestor and evolved by gradual, directional change through diversification. Because bioluminescence in Motyxia has been experimentally demonstrated to be aposematic, forewarning of the animal's cyanide-based toxins, these results are contrary to aposematic theory and empirical evidence that a warning pattern cannot evolve gradually in unpalatable prey. However, gradual evolution of a warning pattern is plausible if faint light emission served another function and was co-opted as an aposematic signal later in the diversification of the genus. Luminescence in Motyxia stem-group taxa may have initially evolved to cope with reactive oxygen stress triggered by a hot, dry environment and was repurposed for aposematism by high-elevation crown-group taxa colonizing new habitats with varying levels of predation. The discovery of bioluminescence in X. bistipita and its pivotal phylogenetic location provides insight into the independent and repeated evolution of bioluminescence across the tree of life.

  2. Increased bioassay sensitivity of bioactive molecule discovery using metal-enhanced bioluminescence

    NASA Astrophysics Data System (ADS)

    Golberg, Karina; Elbaz, Amit; McNeil, Ronald; Kushmaro, Ariel; Geddes, Chris D.; Marks, Robert S.

    2014-12-01

    We report the use of bioluminescence signal enhancement via proximity to deposited silver nanoparticles for bioactive compound discovery. This approach employs a whole-cell bioreporter harboring a plasmid-borne fusion of a specific promoter incorporated with a bioluminescence reporter gene. The silver deposition process was first optimized to provide optimal nanoparticle size in the reaction time dependence with fluorescein. The use of silver deposition of 350 nm particles enabled the doubling of the bioluminescent signal amplitude by the bacterial bioreporter when compared to an untouched non-silver-deposited microtiter plate surface. This recording is carried out in the less optimal but necessary far-field distance. SEM micrographs provided a visualization of the proximity of the bioreporter to the silver nanoparticles. The electromagnetic field distributions around the nanoparticles were simulated using Finite Difference Time Domain, further suggesting a re-excitation of non-chemically excited bioluminescence in addition to metal-enhanced bioluminescence. The possibility of an antiseptic silver effect caused by such a close proximity was eliminated disregarded by the dynamic growth curves of the bioreporter strains as seen using viability staining. As a highly attractive biotechnology tool, this silver deposition technique, coupled with whole-cell sensing, enables increased bioluminescence sensitivity, making it especially useful for cases in which reporter luminescence signals are very weak.

  3. A Causal Relation between Bioluminescence and Oxygen to Quantify the Cell Niche

    PubMed Central

    Lambrechts, Dennis; Roeffaers, Maarten; Goossens, Karel; Hofkens, Johan; Van de Putte, Tom; Schrooten, Jan; Van Oosterwyck, Hans

    2014-01-01

    Bioluminescence imaging assays have become a widely integrated technique to quantify effectiveness of cell-based therapies by monitoring fate and survival of transplanted cells. To date these assays are still largely qualitative and often erroneous due to the complexity and dynamics of local micro-environments (niches) in which the cells reside. Here, we report, using a combined experimental and computational approach, on oxygen that besides being a critical niche component responsible for cellular energy metabolism and cell-fate commitment, also serves a primary role in regulating bioluminescent light kinetics. We demonstrate the potential of an oxygen dependent Michaelis-Menten relation in quantifying intrinsic bioluminescence intensities by resolving cell-associated oxygen gradients from bioluminescent light that is emitted from three-dimensional (3D) cell-seeded hydrogels. Furthermore, the experimental and computational data indicate a strong causal relation of oxygen concentration with emitted bioluminescence intensities. Altogether our approach demonstrates the importance of oxygen to evolve towards quantitative bioluminescence and holds great potential for future microscale measurement of oxygen tension in an easily accessible manner. PMID:24840204

  4. 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...

  5. On the Orientation of Turbulent Structures in Stably Stratified Shear Flows

    NASA Astrophysics Data System (ADS)

    Jacobitz, Frank; Moreau, Adam; Aguirre, Joylene

    2016-11-01

    The orientation of turbulent structures in stably stratified shear flows are investigated using the results of a series of direct numerical simulations. The Richardson number is varied from Ri = 0 , corresponding to unstratified shear flow, to Ri = 1 , corresponding to strongly stratified shear flow. The evolution of the turbulent kinetic energy changes from growth for small Richardson numbers to decay for strong stratification. The orientation of turbulent structures in the flows is determined by the three-dimensional two-point autocorrelation coefficient of velocity magnitude, vorticity magnitude, and fluctuating density. An ellipsoid is fitted to the surface given by a constant autocorrelation coefficient value and the major and minor axes are used to determine the inclination angle of turbulent structures in the plane of shear. The inclination angle is observed to be fairly unaffected by the choice of the autocorrelation coefficient value. In was found that the inclination angle decreases with increasing Richardson number. The structure of the turbulent motion, as characterized by the inclination angle, is therefore directly related to the eventual evolution of the turbulence, as described by the growth or decay rate of the turbulent kinetic energy.

  6. Internal Gravity Wave Fluxes Radiated by a Stably Stratified Turbulent Wake

    NASA Astrophysics Data System (ADS)

    Rowe, Kristopher; Diamessis, Peter

    2016-11-01

    The study of the turbulent wake generated by a bluff body moving through a stably stratified fluid has important applications for naval hydrodynamics as well as geophysical flows around topography. Significant progress has been made in terms of investigating the structure and dynamics of the turbulent wake core and the associated near and far-field spectral properties of the wake-radiated internal gravity wave (IGW) fields, namely in the context of high Reynolds stratified turbulence within the wake itself. Nevertheless, little has been done to quantify the amount of energy and momentum radiated away by the IGWs generated by the wake. Through analysis of a broad Large Eddy Simulation dataset, spanning values of body-based Reynolds and Froude numbers, Re = 5 ×103 ,105 and 4 ×105 and Fr = 4 , 16 and 64, we compute the energy and momentum fluxes of IGWs radiated by the stratified turbulent wake of a towed sphere and explore the relevant parametric dependence. The analysis further aims to determine the potential of the IGWs as a sink for energy and momentum relative to the dissipation of turbulent kinetic energy in the wake itself. Finally, we discuss the implications that for our findings for wake mean-flow self-similarity and turbulence subgrid scale models. Office of Naval Research Grants N00014-13-1-0665 and N00014-15-1-2513.

  7. Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

    NASA Technical Reports Server (NTRS)

    Granger, C. L.; Cyr, R. J.

    2000-01-01

    Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.

  8. [Establishment of stably expressed human RANTES gene in prunella vulgaris cell clone].

    PubMed

    Zeng, Qing-Ping; Feng, Li-Ling; Yang, Rui-Yi; Chen, Zhu-Hua

    2003-03-01

    To express interesting human genes in herbal cells for boosting their specific pharmacological activities, RANTES gene cloned from human peripheral blood lymphocyte (PBL) mRNA was introduced into A. tumefaciens strain LBA4404 harboring pAL4404 plasmid via tumor-inducing (Ti) plasmid-derived intermediate expression vector pROKII. In vitro cultured P. vulgaris cells were transformed by leaf-disk cocultivation procedure. Integration of RANTES gene in the genome of transformed cells was confirmed by Southern blotting, and expression of RANTES gene in transformed cells was analyzed by RT-PCR amplification, Western blotting and enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of PBL was utilized as a detection index of cellular chemotropism induction by recombinant RANTES. The results have shown the RANTES gene was integrated in transgenic P. vulgaris cells, and RANTES gene-stably expressed cell clones were available, which could pave the way to obtain transgenic P. vulgaris plants demonstrating specific pharmacological activities.

  9. Stably superhydrophobic (IL/TiO2)n hybrid films: Intelligent self-cleaning materials

    NASA Astrophysics Data System (ADS)

    Xin, Bingwei; Wang, Limei; Jia, Chunxiao

    2015-12-01

    Stably self-cleaning (IL/TiO2)n nanocomposites were prepared via electrostatic layer-by-layer (LbL) self-assembly technique. Positively charged [C12mim]Br and negatively charged TiO2 nanoparticles were alternatively adsorbed on the negative glass substrates to form (IL/TiO2)n layers. They were characterized by scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS) and UV-vis absorption spectroscopy. Under the synergistic action of ionic liquids and TiO2 P25, in which TiO2 nanoparticles provided surface roughness while [C12mim]Br acted as lower surface tension material, glass coated with 13 bilayers of [C12mim]Br/TiO2 film arrived to superhydrophobicity with 151.7 ± 2°. Owing to the photoresponsive and photocatalytic properties of TiO2, (IL/TiO2)n nanocomposites achieved the reversible superhydrophobic and superhydrophilic transition upon alternating UV irradiation and storage in the dark, and presented good performance for photocatalytic degradation of methyl orange with ultraviolet (UV) illumination. Significantly, they could be recycled for several times without obvious fatigue.

  10. Observation of an internal wave attractor in a confined, stably stratified fluid

    NASA Astrophysics Data System (ADS)

    Maas, Leo R. M.; Benielli, Dominique; Sommeria, Joël; Lam, Frans-Peter A.

    1997-08-01

    When a container of water is vibrated, its response can be described in terms of large-scale standing waves-the eigenmodes of the system. The belief that enclosed continuous media always possess eigenmodes is deeply rooted. Internal gravity waves in uniformly stratified fluids, however, present a counterexample. Such waves propagate at a fixed angle to the vertical that is determined solely by the forcing frequency, and a sloping side wall of the container will therefore act as a lens, resulting in ray convergence or divergence. An important consequence of this geometric focusing is the prediction that, following multiple reflections, these waves will evolve onto specific paths-or attractors-whose locations are determined only by the frequency. Here we report the results of laboratory experiments that confirm that internal-wave attractors, rather than eigenmodes, determine the response of a confined, stably stratified fluid over a broad range of vibration frequencies. The existence of such attractors could be important for mixing processes in ocean basins and lakes, and may be useful for analysing oscillations of the Earth's liquid core and the stability of spinning, fluid-filled spacecraft.

  11. Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells

    PubMed Central

    Mandegar, Mohammad A.; Moralli, Daniela; Khoja, Suhail; Cowley, Sally; Chan, David Y.L.; Yusuf, Mohammed; Mukherjee, Sayandip; Blundell, Michael P.; Volpi, Emanuela V.; Thrasher, Adrian J.; James, William; Monaco, Zoia L.

    2011-01-01

    We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications. PMID:21593218

  12. Dynamics of motile micro-organisms in stably-stratified turbulence

    NASA Astrophysics Data System (ADS)

    Lovecchio, Salvatore; Zonta, Francesco; Marchioli, Cristian; Soldati, Alfredo

    2016-11-01

    Motile micro-organisms populating terrestrial water bodies swim upward towards the air-water interface to capture light and activate photosynthesis. These micro-organisms have the center of mass displaced below the center of buoyancy and are usually called gyrotactic swimmers. Gyrotactic swimmers (which are almost neutrally-buoyant) are extremely sensitive to the local flow field, which is often stably stratified (due to solar heating at the water surface). Stable stratification has a deep influence on the transport processes of mass, momentum, heat and chemical species at the water surface. In this work we use Direct Numerical Simulation (DNS) and Lagrangian Particle Tracking (LPT) to analyze the dynamics of gyrotactic swimmers in stratified turbulence. Our results show that swimmers surfacing and clustering at the surface depend strongly on the re-orientation time of swimmers and on the level of stratification. Obtaining accurate predictions of the surfacing time for gyrotactic swimmers is extremely important to estimate the global CO2 exchange across the air-water interface.

  13. Noise and Turbulence Generate 3D Zombie Vortices in Stably Stratified Rotating Shear Flows

    NASA Astrophysics Data System (ADS)

    Pei, Suyang; Marcus, Philip S.; Jiang, Chung-Hsiang; Hassanzadeh, Pedram; Lecoanet, Daniel; Barranco, Joseph A.

    2013-11-01

    We showed previously that a linearly stable shearing, rotating, stably stratified flow has a finite-amplitude instability creating ``zombie vortices'' that self-replicate and fill the domain. Our flows were initialized with perturbations of one or two vortices. Our motivation was to determine whether ``dead zones'' in protoplanetary disks were stable, or whether they could be de-stabilized to produce vortices necessary for the final part of star formation and for planet formation. To be more relevant to astrophysics, we choose the initial conditions to be noise or turbulence with a Kolmogorov spectrum with small kinetic energy and Mach number. In a Kolmogorov spectrum, the largest eddies determine the kinetic energy and Mach number, while the smallest determine the vorticity and Rossby number Ro ≡ ω / f , where ω is the vertical vorticity and f is the Coriolis parameter. The protoplanetary disks (which have large inertial ranges due to their large Reynolds numbers), can have large Rossby numbers, but weak Mach numbers and kinetic energies. It is important to know whether the triggering of the finite-amplitude instability that creates zombie vortices depends on threshold values of Mach number, kinetic energy, or the Rossby number. Here, we show it is the latter.

  14. Boundary Layer Effects on Internal Wave Generation in a Stably Stratified Fluid

    NASA Astrophysics Data System (ADS)

    Eberly, Lauren; Vanderhoff, Julie

    2010-11-01

    Through a series of laboratory experiments we attempt to quantify internal wave generation due to flow over the rough topography of a continental slope. Although significant progress has been made in flow over rough topography, few experimental studies have been done where the topography is oriented at an angle to both the isobaths and flow. Laboratory investigation is critical as linear theory is not completely accurate in describing generated internal waves. The disparity between linear theory and physical observation is greatest when the wave amplitudes reach a critical level or when boundary layer separation occurs. Previous experimental work on bottom topography suggests that linear theory over predicts the amplitude of generated lee waves as it does not account for effects due to boundary layer separation. This study employs a series of experiments to analyze an approximately two-dimensional, stably stratified fluid undergoing tidal flow over a topographically rough, sloped shelf. The laboratory set up utilizes a corrugated slope towed through the fluid as the forcing mechanism behind internal wave generation. The waves are visualized using the Synthetic Schlieren technique. Results show decreased internal wave amplitude from that predicted by linear theory.

  15. Formation and destabilization of Kelvin-Helmholtz billows in stably stratified turbulence

    NASA Astrophysics Data System (ADS)

    Kimura, Yoshifumi; Herring, Jackson

    2013-11-01

    We investigate the formation and destabilization of Kelvin-Helmholtz billows in stably stratified turbulence using the pseudo-spectral DNS of the Navier-Stokes equations under the Boussinesq approximation with 20483 grid points. Our method is to integrate the equations from the zero total energy initial condition with horizontal forcing imposed in a narrow wave number band. In the course of developments, the horizontal spectra first show a single steep power-law (k ? 4 , 5, where k is the horizontal wavenumber), and then the tail part of the spectrum begins to rise to show the Kolmogorov-type slope (k ? 5 / 3). From the viewpoint of vortex formation, we first observe that many wedge vortices are produced which move horizontally (like dipoles) in random directions. As time goes on, the wings of the wedges become thinner and thinner while translating, and finally detach to be almost independent vortex layers. This thinning mechanism makes the vertical shear stronger and eventually the local Richardson number small enough to produce Kelvin-Helmholtz billows. We will demonstrate that the transition in the horizontal energy spectra has a close relation with the destabilizing process of the Kelvin-Helmholtz billows.

  16. DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

    PubMed Central

    DiFranco, Marino; Quinonez, Marbella; Capote, Joana; Vergara, Julio

    2009-01-01

    A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, β2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and α-actinin; and membrane proteins, i.e. α1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the

  17. DNA transfection of mammalian skeletal muscles using in vivo electroporation.

    PubMed

    DiFranco, Marino; Quinonez, Marbella; Capote, Joana; Vergara, Julio

    2009-10-19

    A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver, skeletal muscle, and even the brain. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP, calpain, FKBP12, beta2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and alpha-actinin; and membrane proteins, i.e. alpha1s-DHPR, RyR1, cardiac Na/Ca(2+) exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical and functional evidence. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the

  18. Dynamic Imaging of Pancreatic Nuclear Factor κB (NF-κB) Activation in Live Mice Using Adeno-associated Virus (AAV) Infusion and Bioluminescence*

    PubMed Central

    Orabi, Abrahim I.; Sah, Swati; Javed, Tanveer A.; Lemon, Kathryn L.; Good, Misty L.; Guo, Ping; Xiao, Xiangwei; Prasadan, Krishna; Gittes, George K.; Jin, Shunqian; Husain, Sohail Z.

    2015-01-01

    Nuclear factor κB (NF-κB) is an important signaling molecule that plays a critical role in the development of acute pancreatitis. Current methods for examining NF-κB activation involve infection of an adenoviral NF-κB-luciferase reporter into cell lines or electrophoretic mobility shift assay of lysate. The use of adeno-associated viruses (AAVs) has proven to be an effective method of transfecting whole organs in live animals. We examined whether intrapancreatic duct infusion of AAV containing an NF-κB-luciferase reporter (AAV-NF-κB-luciferase) can reliably measure pancreatic NF-κB activation. We confirmed the infectivity of the AAV-NF-κB-luciferase reporter in HEK293 cells using a traditional luciferase readout. Mice were infused with AAV-NF-κB-luciferase 5 weeks before induction of pancreatitis (caerulein, 50 μg/kg). Unlike transgenic mice that globally express NF-κB-luciferase, AAV-infused mice showed a 15-fold increase in pancreas-specific NF-κB bioluminescence following 12 h of caerulein compared with baseline luminescence (p < 0.05). The specificity of the NF-κB-luciferase signal to the pancreas was confirmed by isolating the pancreas and adjacent organs and observing a predominant bioluminescent signal in the pancreas compared with liver, spleen, and stomach. A complementary mouse model of post-ERCP-pancreatitis also induced pancreatic NF-κB signals. Taken together these data provide the first demonstration that NF-κB activation can be examined in a live, dynamic fashion during pancreatic inflammation. We believe this technique offers a valuable tool to study real-time activation of NF-κB in vivo. PMID:25802340

  19. Collision of millimetre droplets induces DNA and protein transfection into cells

    PubMed Central

    Ikemoto, Kazuto; Sakata, Ichiro; Sakai, Takafumi

    2012-01-01

    Nonperturbing and simple transfection methods are important for modern techniques used in biotechnology. Recently, we reported that electrospraying can be applied to DNA transfection in cell lines, bacteria, and chicken embryos. However, the transfection efficiency was only about 2%. To improve the transfection rate, physical properties of the sprayed droplets were studied in different variations of the method. We describe a highly efficient technique (30–93%) for introduction of materials such as DNA and protein into living cells by electrospraying droplets of a high conductivity liquid onto cells incubated with the material for transfection. Electric conductivity has a sizable influence on the success of transfection. In contrast, molecular weight of the transfected material, types of ions in the electrospray solution, and the osmotic pressure do not influence transfection efficiency. The physical analysis revealed that collision of cells with millimetre-sized droplets activates intracellular uptake. PMID:22375250

  20. Humic acid inhibits HBV-induced autophagosome formation and induces apoptosis in HBV-transfected Hep G2 cells

    PubMed Central

    Pant, Kishor; Yadav, Ajay K.; Gupta, Parul; Rathore, Abhishek Singh; Nayak, Baibaswata; Venugopal, Senthil K.

    2016-01-01

    Hepatitis B Virus (HBV) utilizes several mechanisms to survive in the host cells and one of the main pathways being autophagosome formation. Humic acid (HA), one of the major components of Mineral pitch, is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. We hypothesized that HA could induce cell death and inhibit HBV-induced autophagy in hepatic cells. Incubation of Hep G2.2.1.5 cells (HepG2 cells stably expressing HBV) with HA (100 μM) inhibited both cell proliferation and autophagosome formation significantly, while apoptosis induction was enhanced. Western blot results showed that HA incubation resulted in decreased levels of beclin-1, SIRT-1 and c-myc, while caspase-3 and β-catenin expression were up-regulated. Western blot results showed that HA significantly inhibited the expression of HBx (3-fold with 50 μM and 5-fold with 100 μM) compared to control cells. When HA was incubated with HBx-transfected Hep G2 cells, HBx-induced autophagosome formation and beclin-1 levels were decreased. These data showed that HA induced apoptosis and inhibited HBV-induced autophagosome formation and proliferation in hepatoma cells. PMID:27708347

  1. Delayed expression of apoptosis in X-irradiated human leukemic MOLT-4 cells transfected with mutant p53.

    PubMed

    Nakano, Hisako; Yonekawa, Hiromichi; Shinohara, Kunio

    2003-06-01

    The effects of X-rays on cell survival, apoptosis, and long-term response in the development of cell death as measured by the dye exclusion test were studied in human leukemic MOLT-4 cells (p53 wild-type) stably transfected with a mutant p53 cDNA expression vector. Cell survival, as determined from colony-forming ability, was increased in an expression level dependent manner, but the increase was partial even with the highest-expressing clone (B3). This contrasts with the prior observation that cell death and apoptosis in B3 are completely inhibited at 24 h after irradiation with 1.8 Gy of X-rays. The examination of B3 cells incubated for longer than 24 h after X-irradiation showed a delay in the induction of cell death and apoptosis. Western blot analysis revealed that the time required to reach the highest level of wild-type p53 protein in B3 was longer than the time in MOLT-4 and that the p53 may be stabilized by the phosphorylation at Ser-15. These results suggest that the introduction of mutant p53 into MOLT-4 merely delays the development of apoptosis, during which the cells could repair the damage induced by X-rays, and results in the partial increase in cell survival.

  2. PUMA gene transfection can enhance the sensitivity of epirubicin-induced apoptosis of MCF-7 breast cancer cells.

    PubMed

    Sun, C-G; Zhuang, J; Teng, W-J; Wang, Z; Du, S-S

    2015-05-29

    We explored whether p53 upregulated modulator of apoptosis (PUMA) gene transfection could enhance the sensitivity of epirubicin-induced apoptosis of MCF-7 breast cancer cells. The liposome-mediated recombinant eukaryotic expression vector PU-MA-pCDNA3 and empty vector plasmid were stably transfected into MCF-7 cells. Epirubicin (0.01-100 μM) was applied to MCF-7, MCF-7/PUMA, and MCF-7/pCDNA3 cells for 72 h. The MTT assay was used to calculate the cell survival rate in each group, and the 50% inhibitory concentration (IC50) was calculated. The IC50 values of epirubicin in MCF-7, MCF-7/PUMA, and MCF-7/pCDNA3 cells were 13 ± 1.4, 1.8 ± 0.2, and 10.7 ± 1.3 μM, respectively. The sensitivity of MCF-7/PUMA cells to epirubicin increased 7.2-fold. Epirubicin induced apoptosis in MCF-7 cells dose-dependently, but MCF-7/PUMA cell-induced apoptosis was more significant compared to controls. Low concentrations of epirubicin (0.1 μM) caused low levels of apoptosis of MCF-7/pCDNA3 (1.15 ± 0.26%) and MCF-7 cells (0.9 ± 0.24%), but significantly induced apoptosis of MCF-7/PUMA cells (6.44 ± 1.46%). High epirubicin concentration (1 μM) induced apoptosis in each group, but the epirubicin MCF-7/PUMA apoptosis rate (35.47 ± 9.36%) was significantly higher than that of MCF-7 (12.6 ± 3.73%) and MCF-7/ pCDNA3 (15.2 ± 5.17%) cells (P < 0 01). Flow cytometry and TUNEL assays for apoptosis detection showed similar results. PUMA protein expression in MCF-7/PUMA cells was significantly higher than that in MCF-7 and MCF-7/pCDNA3 cells by Western blot analysis. There-fore, stable transfection of PUMA can significantly enhance epirubicin-induced apoptosis sensitivity of MCF-7 breast cancer cells.

  3. Molecular Detection of Bioluminescent Dinoflagellates in Surface Waters of the Patagonian Shelf during Early Austral Summer 2008

    PubMed Central

    Valiadi, Martha; Painter, Stuart C.; Allen, John T.; Balch, William M.; Iglesias-Rodriguez, M. Debora

    2014-01-01

    We investigated the distribution of bioluminescent dinoflagellates in the Patagonian Shelf region using “universal” PCR primers for the dinoflagellate luciferase gene. Luciferase gene sequences and single cell PCR tests, in conjunction with taxonomic identification by microscopy, allowed us to identify and quantify bioluminescent dinoflagellates. We compared these data to coincidental discrete optical measurements of stimulable bioluminescence intensity. Molecular detection of the luciferase gene showed that bioluminescent dinoflagellates were widespread across the majority of the Patagonian Shelf region. Their presence was comparatively underestimated by optical bioluminescence measurements, whose magnitude was affected by interspecific differences in bioluminescence intensity and by the presence of other bioluminescent organisms. Molecular and microscopy data showed that the complex hydrography of the area played an important role in determining the distribution and composition of dinoflagellate populations. Dinoflagellates were absent south of the Falkland Islands where the cold, nutrient-rich, and well-mixed waters of the Falklands Current favoured diatoms instead. Diverse populations of dinoflagellates were present in the warmer, more stratified waters of the Patagonian Shelf and Falklands Current as it warmed northwards. Here, the dinoflagellate population composition could be related to distinct water masses. Our results provide new insight into the prevalence of bioluminescent dinoflagellates in Patagonian Shelf waters and demonstrate that a molecular approach to the detection of bioluminescent dinoflagellates in natural waters is a promising tool for ecological studies of these organisms. PMID:24918444

  4. In vivo imaging of mice infected with bioluminescent Trypanosoma cruzi unveils novel sites of infection

    PubMed Central

    2014-01-01

    Background The development of techniques that allow the imaging of animals infected with parasites expressing luciferase opens up new possibilities for following the fate of parasites in infected mammals. Methods D-luciferin potassium salt stock solution was prepared in phosphate-buffered saline (PBS) at 15 mg/ml. To produce bioluminescence, infected and control mice received an intraperitoneal injection of luciferin stock solution (150 mg/kg). All mice were immediately anesthetized with 2% isofluorane, and after 10 minutes were imaged. Ex vivo evaluation of infected tissues and organs was evaluated in a 24-well plate in 150 μg/ml D-luciferin diluted in PBS. Images were captured using the IVIS Lumina image system (Xenogen). Dissected organs were also evaluated by microscopy of hematoxylin-eosin stained sections. Results Here we describe the results obtained using a genetically modified Dm28c strain of T. cruzi expressing the firefly luciferase to keep track of infection by bioluminescence imaging. Progression of infection was observed in vivo in BALB/c mice at various intervals after infection with transgenic Dm28c-luc. The bioluminescent signal was immediately observed at the site of T. cruzi inoculation, and one day post infection (dpi) it was disseminated in the peritoneal cavity. A similar pattern in the cavity was observed on 7 dpi, but the bioluminescence was more intense in the terminal region of the large intestine, rectum, and gonads. On 14 and 21 dpi, bioluminescent parasites were also observed in the heart, snout, paws, hind limbs, and forelimbs. From 28 dpi to 180 dpi in chronically infected mice, bioluminescence declined in regions of the body but was concentrated in the gonad region. Ex vivo evaluation of dissected organs and tissues by bioluminescent imaging confirmed the in vivo bioluminescent foci. Histopathological analysis of dissected organs demonstrated parasite nests at the rectum and snout, in muscle fibers of mice infected with Dm28c

  5. Delivery of episomal vectors into primary cells by means of commercial transfection reagents.

    PubMed

    Han, Na Rae; Lee, Hyun; Baek, Song; Yun, Jung Im; Park, Kyu Hyun; Lee, Seung Tae

    2015-05-29

    Although episomal vectors are commonly transported into cells by electroporation, a number of electroporation-derived problems have led to the search for alternative transfection protocols, such as the use of transfection reagents, which are inexpensive and easy to handle. Polyplex-mediated transport of episomal vectors into the cytoplasm has been conducted successfully in immortalized cell lines, but no report exists of successful transfection of primary cells using this method. Accordingly, we sought to optimize the conditions for polyplex-mediated transfection for effective delivery of episomal vectors into the cytoplasm of primary mouse embryonic fibroblasts. Episomal vectors were complexed with the commercially available transfection reagents Lipofectamine 2000, FuGEND HD and jetPEI. The ratio of transfection reagent to episomal vectors was varied, and the subsequent transfection efficiency and cytotoxicity of the complexes were analyzed using flow cytometry and trypan blue exclusion assay, respectively. No cytotoxicity and the highest transfection yield were observed when the ratio of transfection reagent to episomal vector was 4 (v/wt) in the cases of Lipofectamine 2000 and FuGENE HD, and 2 in the case of jetPEI. Of the three transfection reagents tested, jetPEI showed the highest transfection efficiency without any cytotoxicity. Thus, we confirmed that the transfection reagent jetPEI could be used to effectively deliver episomal vectors into primary cells without electroporation.

  6. A new bioluminescent cellular assay to measure the transcriptional effects of chemicals that modulate the alpha-1 thyroid hormone receptor.

    PubMed

    Jugan, M L; Lévy-Bimbot, M; Pomérance, M; Tamisier-Karolak, S; Blondeau, J P; Lévi, Y

    2007-09-01

    Interactions of environmental pollutants with the thyroid endocrine axis have received much attention especially because thyroid hormones (THs) play a major role in mammalian brain development. In order to screen for compounds that act on the triiodothyronine (T3) signaling pathway, we developed a new reporter gene assay expressing luciferase under the control of the TH receptor (TR). PC12 cells expressing the alpha1-isoform of TR of avian origin were stably transfected with a luciferase gene controlled by the SV40 promoter, and enhanced by a four-spaced direct repeat (DR4) thyroid response element (TRE). The resulting PC-DR-LUC cells were used to optimize a T3 assay in multiwell microplates. This assay was highly sensitive (30 pM T3) and reproducible, and responded as expected to TH analogues. Several halogenated phenolic (3,3',5,5'-tetrabromobisphenol A, 3,3',5,5'-tetrachlorobisphenol A, 4-hydroxy-2',3,4',5,6'-pentachlorobiphenyl) and phenol (pentachlorophenol, 2,4,6-triiodophenol) compounds suspected of being thyroid-disrupting environmental chemicals induced partial agonistic and/or complex competitive/uncompetitive antagonistic responses in PC-DR-LUC cells at micromolar concentrations. A cell viability test indicated that these effects were not related to cytotoxicity of the chemicals. These results suggest that the PC-DR-LUC assay could be a valuable tool for the large-scale screening for thyroid receptor agonists and antagonists in vitro, and for detecting thyroid disruptors in the environment.

  7. The eddy, wave, and interface structure of turbulent shear layers below/above stably stratified regions

    NASA Astrophysics Data System (ADS)

    Hunt, Julian C. R.; Moustaoui, Mohamed; Mahalov, Alex

    2015-09-01

    High resolution three-dimensional simulations are presented of the interactions between turbulent shear flows moving with mean relative velocity ΔU below a stably stratified region with buoyancy frequency (N+). An artificial forcing in the simulation, with a similar effect as a small negative eddy viscosity, leads to a steady state flow which models thin interfaces. Characteristic eddies of the turbulence have length scale L. If the bulk Richardson number Rib=(LN+/ΔU)2 lies between lower and upper critical values denoted as Ri∗(<1/5) and R~i(˜ 1), a "detached" layer is formed in the stable region with thickness L+ greater than L, in which rotational fluctuations and inhomogeneous turbulence are induced above an interface with large gradients of density/temperature. Comparisons are made with shear turbulent interfaces with no stratification. When Rib>R~i, vertical propagating waves are generated, with shear stresses carrying significant momentum flux and progressively less as Rib increases. Simulations for a jet and a turbulent mixing layer show similar results. A perturbation analysis, using inhomogeneous Rapid Distortion Theory, models the transition zone between shear eddies below the interface and the fluctuations in the stratified region, consistent with the simulations. It demonstrates how the wave-momentum-flux has a maximum when Rib˜2 and then decreases as Rib increases. This coupling mechanism between eddies and waves, which is neglected in eddy viscosity models for shear layers, can drive flows in the stratosphere and the deeper ocean, with significant consequences for short- and long-term flow phenomena. The "detached layer" is a mechanism that contributes to the formation of stratus clouds and polluted layers above the atmospheric boundary layer.

  8. Phenomenology of two-dimensional stably stratified turbulence under large-scale forcing

    NASA Astrophysics Data System (ADS)

    Kumar, Abhishek; Verma, Mahendra K.; Sukhatme, Jai

    2017-03-01

    In this paper we characterize the scaling of energy spectra, and the interscale transfer of energy and enstrophy, for strongly, moderately and weakly stably stratified two-dimensional (2D) turbulence under large-scale random forcing. In the strongly stratified case, a large-scale vertically sheared horizontal flow (VSHF) co-exists with small scale turbulence. The VSHF consists of internal gravity waves and the turbulent flow has a kinetic energy (KE) spectrum that follows an approximate $k^{-3}$ scaling with zero KE flux and a robust positive enstrophy flux. The spectrum of the turbulent potential energy (PE) also approximately follows a $k^{-3}$ power-law and its flux is directed to small scales. For moderate stratification, there is no VSHF and the KE of the turbulent flow exhibits Bolgiano-Obukhov scaling that transitions from a shallow $k^{-11/5}$ form at large scales, to a steeper approximate $k^{-3}$ scaling at small scales. The entire range of scales shows a strong forward enstrophy flux, and interestingly, large (small) scales show an inverse (forward) KE flux. The PE flux in this regime is directed to small scales, and the PE spectrum is characterized by an approximate $k^{-1.64}$ scaling. Finally, for weak stratification, KE is transferred upscale and its spectrum closely follows a $k^{-2.5}$ scaling, while PE exhibits a forward transfer and its spectrum shows an approximate $k^{-1.6}$ power-law. For all stratification strengths, the total energy always flows from large to small scales and almost all the spectral indices are well explained by accounting for the scale dependent nature of the corresponding flux.

  9. Stem cells expanded from the human embryonic hindbrain stably retain regional specification and high neurogenic potency.

    PubMed

    Tailor, Jignesh; Kittappa, Raja; Leto, Ketty; Gates, Monte; Borel, Melodie; Paulsen, Ole; Spitzer, Sonia; Karadottir, Ragnhildur Thora; Rossi, Ferdinando; Falk, Anna; Smith, Austin

    2013-07-24

    Stem cell lines that faithfully maintain the regional identity and developmental potency of progenitors in the human brain would create new opportunities in developmental neurobiology and provide a resource for generating specialized human neurons. However, to date, neural progenitor cultures derived from the human brain have either been short-lived or exhibit restricted, predominantly glial, differentiation capacity. Pluripotent stem cells are an alternative source, but to ascertain definitively the identity and fidelity of cell types generated solely in vitro is problematic. Here, we show that hindbrain neuroepithelial stem (hbNES) cells can be derived and massively expanded from early human embryos (week 5-7, Carnegie stage 15-17). These cell lines are propagated in adherent culture in the presence of EGF and FGF2 and retain progenitor characteristics, including SOX1 expression, formation of rosette-like structures, and high neurogenic capacity. They generate GABAergic, glutamatergic and, at lower frequency, serotonergic neurons. Importantly, hbNES cells stably maintain hindbrain specification and generate upper rhombic lip derivatives on exposure to bone morphogenetic protein (BMP). When grafted into neonatal rat brain, they show potential for integration into cerebellar development and produce cerebellar granule-like cells, albeit at low frequency. hbNES cells offer a new system to study human cerebellar specification and development and to model diseases of the hindbrain. They also provide a benchmark for the production of similar long-term neuroepithelial-like stem cells (lt-NES) from pluripotent cell lines. To our knowledge, hbNES cells are the first demonstration of highly expandable neuroepithelial stem cells derived from the human embryo without genetic immortalization.

  10. Turbulent mixing in stably stratified flows of the environment: The current state of the problem (Review)

    NASA Astrophysics Data System (ADS)

    Vasil'Ev, O. F.; Voropaeva, O. F.; Kurbatskii, A. F.

    2011-06-01

    Specific features of the turbulent transfer of the momentum and heat in stably stratified geophysical flows, as well as possibilities for including them into RANS turbulence models, are analyzed. The momentum (but not heat) transfer by internal gravity waves under conditions of strong stability is, for example, one such feature. Laboratory data and measurements in the atmosphere fix a clear dropping trend of the inverse turbulent Prandtl number with an increasing gradient Richardson number, which must be reproduced by turbulence models. Ignoring this feature can cause a false diffusion of heat under conditions of strong stability and lead, in particular, to noticeable errors in calculations of the temperature in the atmospheric boundary layer. Therefore, models of turbulent transfer must include the effect of the action of buoyancy and internal gravity waves on turbulent flows of the momentum. Such a strategy of modeling the stratified turbulence is presented in the review by a concrete RANS model and original results obtained during the modeling of stratified flows in the environment. Semiempirical turbulence models used for calculations of complex turbulent flows in deep stratified bodies of water are also analyzed. This part of the review is based on the data of investigations within the framework of the large international scientific Comparative Analysis and Rationalization of Second-Moment Turbulence Models (CARTUM) project and other publications of leading specialists. The most economical and effective approach associated with modified two-parameter turbulence models is a real alternative to classical variants of these models. A class of test problems and laboratory and full-scale experiments used by the participants of the CARTUM project for the approbation of numerical models are considered.

  11. Stably stratified shear turbulence: A new model for the energy dissipation length scale

    NASA Technical Reports Server (NTRS)

    Cheng, Y.; Canuto, V. M.

    1994-01-01

    A model is presented to compute the turbulent kinetic energy dissipation length scale l(sub epsilon) in a stably stratified shear flow. The expression for l(sub epsilon) is derived from solving the spectral balance equation for the turbulent kinetic energy. The buoyancy spectrum entering such equation is constructed using a Lagrangian timescale with modifications due to stratification. The final result for l(sub epsilon) is given in algebraic form as a function of the Froude number Fr and the flux Richardson number R(sub f), l(sub epsilon) = l(sub epsilon)(Fr, R(sub f). The model predicts that for R(sub f) less than R(sub fc), l(sub epsilon) decreases with stratification. An attractive feature of the present model is that it encompasses, as special cases, some seemingly different models for l(sub epsilon) that have been proposed in the past by Deardorff, Hunt et al., Weinstock, and Canuto and Minotti. An alternative form for the dissipation rate epsilon is also discussed that may be useful when one uses a prognostic equation for the heat flux. The present model is applicable to subgrid-scale models, which are needed in large eddy simulations (LES), as well as to ensemble average models. The model is applied to predict the variation of l(sub epsilon) with height z in the planetary boundary layer. The resulting l(sub epsilon) versus z profile reproduces very closely the nonmonotonic profile of l(sub epsilon) exhibited by many LES calculations, beginning with the one by Deardorff in 1974.

  12. Interactions between gravity waves and cold air outflows in a stably stratified uniform flow

    NASA Technical Reports Server (NTRS)

    Lin, Yuh-Lang; Wang, Ting-An; Weglarz, Ronald P.

    1993-01-01

    Interactions between gravity waves and cold air outflows in a stably stratified uniform flow forced by various combinations of prescribed heat sinks and sources are studied using a hydrostatic two-dimensional nonlinear numerical model. The formation time for the development of a stagnation point or reversed flow at the surface is not always directly proportional to the Froude number when wave reflections exist from upper levels. A density current is able to form by the wave-otuflow interaction, even though the Froude number is greater than a critical value. This is the result of the wave-outflow interaction shifting the flow response to a different location in the characteristic parameter space. A density current is able to form or be destroyed due to the wave-outflow interaction between a traveling gravity wave and cold air outflow. This is proved by performing experiments with a steady-state heat sink and an additional transient heat source. In a quiescent fluid, a region of cold air, convergence, and upward motion is formed after the collision between two outflows produced by two prescribed heat sinks. After the collision, the individual cold air outflows lose their own identity and merge into a single, stationary, cold air outflow region. Gravity waves tend to suppress this new stationary cold air outflow after the collision. The region of upward motion associated with the collision is confined to a very shallow layer. In a moving airstream, a density current produced by a heat sink may be suppressed or enhanced nonlinearly by an adjacent heat sink due to the wave-outflow interaction.

  13. Direct numerical simulations of stably-stratified sheared turbulence: Implications for oceanic mixing

    NASA Technical Reports Server (NTRS)

    Itsweire, E. C.; Holt, S. E.; Koseff, J. R.; Ferziger, J. H.

    1990-01-01

    Direct numerical simulations of the time evolution of homogeneous stably stratified turbulent shear flows have been performed for several Richardson numbers Ri and Reynolds numbers R(sub lambda) in earlier works. The results show excellent agreement with length scale models developed from laboratory experiments to characterize oceanic turbulence. When the Richardson number Ri is less than the stationary value Ri(sub s), the turbulence intensity grows at all scales, and the growth rate appears to be a function of Ri. The size of the vertical density inversions also increases. On the other hand, when Ri is greater than or equal to Ri(sub s) the largest turbulent eddies become vertically constrained by buoyancy when the Ellison (turbulence) scale L(sub E) and the Ozmidov (buoyancy) scale L(sub O) are equal. At this point, the mixing efficiency is maximal and corresponds to a flux Richardson number R(sub f) = 0.20. The vertical mass flux becomes counter-gradient when epsilon = 19(nu)N(exp 2) and vertical density overturns are suppressed in less than half a Brunt-Vaisala period. The results of the simulations were also recast in terms of the Hydrodynamic Phase Diagram introduced in fossil turbulence models. The so-called point of fossilization occurs when epsilon = 4DCN(exp 2); Gibson proposed 13DCN(exp 2). This value is in agreement with indirect laboratory observations and field observations. Finally, the validity of the steady-state models to estimate vertical eddy diffusivities in the oceanic thermocline is discussed.

  14. The origins of marine bioluminescence: turning oxygen defence mechanisms into deep-sea communication tools.

    PubMed

    Rees, J F; de Wergifosse, B; Noiset, O; Dubuisson, M; Janssens, B; Thompson, E M

    1998-04-01

    Bioluminescence, the emission of ecologically functional light by living organisms, emerged independently on several occasions, yet the evolutionary origins of most bioluminescent systems remain obscure. We propose that the luminescent substrates of the luminous reactions (luciferins) are the evolutionary core of most systems, while luciferases, the enzymes catalysing the photogenic oxidation of the luciferin, serve to optimise the expression of the endogenous chemiluminescent properties of the luciferin. Coelenterazine, a luciferin occurring in many marine bioluminescent groups, has strong antioxidative properties as it is highly reactive with reactive oxygen species such as the superoxide anion or peroxides. We suggest that the primary function of coelenterazine was originally the detoxification of the deleterious oxygen derivatives. The functional shift from its antioxidative to its light-emitting function might have occurred when the strength of selection for antioxidative defence mechanisms decreased. This might have been made possible when marine organisms began colonising deeper layers of the oceans, where exposure to oxidative stress is considerably reduced because of reduced light irradiance and lower oxygen levels. A reduction in metabolic activity with increasing depth would also have decreased the endogenous production of reactive oxygen species. Therefore, in these organisms, mechanisms for harnessing the chemiluminescence of coelenterazine in specialised organs could have developed, while the beneficial antioxidative properties were maintained in other tissues. The full range of graded irradiance in the mesopelagic zone, where the majority of organisms are bioluminescent, would have provided a continuum for the selection and improvement of proto-bioluminescence. Although the requirement for oxygen or reactive oxygen species observed in bioluminescent systems reflects the high energy required to produce visible light, it may suggest that oxygen

  15. U-SPECT-BioFluo: an integrated radionuclide, bioluminescence, and fluorescence imaging platform

    PubMed Central

    2014-01-01

    Background In vivo bioluminescence, fluorescence, and single-photon emission computed tomography (SPECT) imaging provide complementary information about biological processes. However, to date these signatures are evaluated separately on individual preclinical systems. In this paper, we introduce a fully integrated bioluminescence-fluorescence-SPECT platform. Next to an optimization in logistics and image fusion, this integration can help improve understanding of the optical imaging (OI) results. Methods An OI module was developed for a preclinical SPECT system (U-SPECT, MILabs, Utrecht, the Netherlands). The applicability of the module for bioluminescence and fluorescence imaging was evaluated in both a phantom and in an in vivo setting using mice implanted with a 4 T1-luc + tumor. A combination of a fluorescent dye and radioactive moiety was used to directly relate the optical images of the module to the SPECT findings. Bioluminescence imaging (BLI) was compared to the localization of the fluorescence signal in the tumors. Results Both the phantom and in vivo mouse studies showed that superficial fluorescence signals could be imaged accurately. The SPECT and bioluminescence images could be used to place the fluorescence findings in perspective, e.g. by showing tracer accumulation in non-target organs such as the liver and kidneys (SPECT) and giving a semi-quantitative read-out for tumor spread (bioluminescence). Conclusions We developed a fully integrated multimodal platform that provides complementary registered imaging of bioluminescent, fluorescent, and SPECT signatures in a single scanning session with a single dose of anesthesia. In our view, integration of these modalities helps to improve data interpretation of optical findings in relation to radionuclide images. PMID:25386389

  16. Tissue Engineering Using Transfected Growth-Factor Genes

    NASA Technical Reports Server (NTRS)

    Madry, Henning; Langer, Robert S.; Freed, Lisa E.; Trippel, Stephen; Vunjak-Novakovic, Gordana

    2005-01-01

    A method of growing bioengineered tissues includes, as a major component, the use of mammalian cells that have been transfected with genes for secretion of regulator and growth-factor substances. In a typical application, one either seeds the cells onto an artificial matrix made of a synthetic or natural biocompatible material, or else one cultures the cells until they secrete a desired amount of an extracellular matrix. If such a bioengineered tissue construct is to be used for surgical replacement of injured tissue, then the cells should preferably be the patient s own cells or, if not, at least cells matched to the patient s cells according to a human-leucocyteantigen (HLA) test. The bioengineered tissue construct is typically implanted in the patient's injured natural tissue, wherein the growth-factor genes enhance metabolic functions that promote the in vitro development of functional tissue constructs and their integration with native tissues. If the matrix is biodegradable, then one of the results of metabolism could be absorption of the matrix and replacement of the matrix with tissue formed at least partly by the transfected cells. The method was developed for articular chondrocytes but can (at least in principle) be extended to a variety of cell types and biocompatible matrix materials, including ones that have been exploited in prior tissue-engineering methods. Examples of cell types include chondrocytes, hepatocytes, islet cells, nerve cells, muscle cells, other organ cells, bone- and cartilage-forming cells, epithelial and endothelial cells, connective- tissue stem cells, mesodermal stem cells, and cells of the liver and the pancreas. Cells can be obtained from cell-line cultures, biopsies, and tissue banks. Genes, molecules, or nucleic acids that secrete factors that influence the growth of cells, the production of extracellular matrix material, and other cell functions can be inserted in cells by any of a variety of standard transfection techniques.

  17. Graphene for improved femtosecond laser based pluripotent stem cell transfection.

    PubMed

    Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Khanyile, Thulile; Warner, Jamie H

    2014-05-01

    Pluripotent stem cells are hugely attractive in the tissue engineering research field as they can self-renew and be selectively differentiated into various cell types. For stem cell and tissue engineering research it is important to develop new, biocompatible scaffold materials and graphene has emerged as a promising material in this area as it does not compromise cell proliferation and accelerates specific cell differentiation. Previous studies have shown a non-invasive optical technique for mouse embryonic stem (mES) cell differentiation and transfection using femtosecond (fs) laser pulses. To investigate cellular responses to the influence of graphene and laser irradiation, here we present for the first time a study of mES cell fs laser transfection on graphene coated substrates. First we studied the impact of graphene on Chinese Hamster Ovary (CHO-K1) cell viability and cell cytotoxicity in the absence of laser exposure. These were tested via evaluating the mitochondrial activity through adenosine triphosphates (ATP) luminescence and breakages on the cell plasma membrane assessed using cytosolic lactate dehydrogenase (LDH) screening. Secondly, the effects of fs laser irradiation on cell viability and cytotoxicity at 1064 and 532 nm for cells plated and grown on graphene and pure glass were assessed. Finally, optical transfection of CHO-K1 and mES cells was performed on graphene coated versus plain glass substrates. Our results show graphene stimulated cell viability whilst triggering a mild release of intracellular LDH. We also observed that compared to pure glass substrates; laser irradiation at 1064 nm on graphene plates was less cytotoxic. Finally, in mES cells efficient optical transfection at 1064 (82%) and 532 (25%) nm was obtained due to the presence of a graphene support as compared to pristine glass. Here we hypothesize an up-regulation of cell adhesion promoting peptides or laminin-related receptors of the extracellular matrix (ECM) in cell samples

  18. A Panel of Trypanosoma brucei Strains Tagged with Blue and Red-Shifted Luciferases for Bioluminescent Imaging in Murine Infection Models

    PubMed Central

    Van Reet, Nick; Van de Vyver, Hélène; Pyana, Patient Pati; Van der Linden, Anne Marie; Büscher, Philippe

    2014-01-01

    Background Genetic engineering with luciferase reporter genes allows monitoring Trypanosoma brucei (T.b.) infections in mice by in vivo bioluminescence imaging (BLI). Until recently, luminescent T.b. models were based on Renilla luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients. Methodology/Principal findings We transfected T.b. brucei, T.b. rhodesiense and T.b. gambiense strains with either RLuc, click beetle red (CBR) or Photinus pyralis RE9 (PpyRE9) luciferase and characterised their in vitro luciferase activity, growth profile and drug sensitivity, and their potential for in vivo BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of T.b. brucei AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged T.b. gambiense LiTaR1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during in vivo BLI. Ex vivo BLI on the brain confirmed central nervous system infection by all luminescent strains of T.b. brucei AnTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT. Conclusions/Significance We established a genetically and phenotypically diverse collection of bioluminescent T.b. brucei, T.b. gambiense and T.b. rhodesiense strains, including drug resistant strains. For in vivo BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity in vivo and at the same time they improve the spatial resolution of the parasites in the entire body due to the better

  19. Co-Registration of Bioluminescence Tomography, Computed Tomography, and Magnetic Resonance Imaging for Multimodal In Vivo Stem Cell Tracking

    PubMed Central

    Chehade, Moussa; Srivastava, Amit K.; Bulte, Jeff W.M.

    2016-01-01

    We present a practical approach for co-registration of bioluminescence tomography (BLT), computed tomography (CT), and magnetic resonance (MR) images. To this end, we developed a customized animal shuttle composed of non-fluorescent, MR-compatible Delrin plastic that fits a commercially available MR surface coil. Mouse embryonic stem cells (mESCs) were transfected with the luciferase gene and labeled with superparamagnetic iron oxide (SPIO) nanoparticles. Cells were stereotaxically implanted in mouse brain and imaged weekly for 4 weeks with BLI (IVIS Spectrum CT scanner) and MRI (11.7T horizontal bore scanner). Without the use of software co-registration, in vitro phantom studies yielded root-mean-square errors (RMSE) of 7.6×10−3, 0.93 mm, and 0.78 mm along the medial-lateral (ML), dorsal-ventral (DV), and anterior-posterior (AP) axes, respectively. Rotation errors were negligible. Software co-registration by translation along the DV and AP axes resulted in consistent agreement between the CT and MR images, without the need for rotation or warping. In vivo co-registered BLT/MRI mouse brain data sets demonstrated a single, diffuse region of BLI photon signal and MRI hypointensity. Over time, the transplanted cells formed tumors as validated by histopathology. Disagreement between BLT and MRI tumor location was greatest along the DV axis (1.4±0.2 mm) compared to the ML (0.5±0.3 mm) and AP axis (0.6 mm) due to the uncertainty of the depth of origin of the BLT signal. Combining the high spatial anatomical information of MRI with the cell viability/proliferation data from BLT should facilitate pre-clinical evaluation of novel therapeutic candidate stem cells. PMID:27478872

  20. Bioluminescent imaging of bacterial biofilm infections in vivo.

    PubMed

    Kadurugamuwa, Jagath L; Francis, Kevin P

    2008-01-01

    Whole body biophotonic imaging (BPI) is a technique that has contributed significantly to the way researchers study bacterial pathogens and develop pre-clinical treatments to combat their ensuing infections in vivo. Not only does this approach allow disease profiles and drug efficacy studies to be conducted non-destructively in live animals over the entire course of the disease, but in many cases, it enables investigators to observe disease profiles that could otherwise easily be missed using conventional methodologies. The principles of this technique are that bacterial pathogens engineered to express bioluminescence (visible light) can be readily monitored from outside of the living animal using specialized low-light imaging equipment, enabling their movement, expansion and treatment to be seen completely non-invasively. Moreover, because the same group of animals can be imaged at each time-point throughout the study, the overall number of animals used is dramatically reduced, saving lives, time, and money. Also, as each animal acts as its own control over time, the issues associated with animal-to-animal variation are circumvented, thus improving the quality of the biostatistical data generated. The ability to monitor infections in vivo in a longitudinal fashion is especially appealing to assess chronic infections such as those involving implanted devices. Typically, bacteria grow as biofilms on these foreign bodies and are reputably difficult to monitor with conventional methods. Because of the non-destructive and non-invasive nature of BPI, the procedure can be performed repeatedly in the same animal, allowing the biofilm to be studied in situ without detachment or disturbance. This ability not only allows unique patterns of disease relapse to be seen following termination of antibiotic therapy but also in vivo resistance development during prolonged treatment, both of which are common occurrences with device-related infections. This chapter describes the

  1. Expression Profiling of a Human Thyroid Cell Line Stably Expressing the BRAFVV600E Mutation

    PubMed Central

    KIM*, BYOUNG-AE; JEE*, HYEON-GUN; WOOK YI*, JIN; KIM, SU-JIN; JUN CHAI, YOUNG; YOUNG CHOI, JUNE; EUN LEE, KYU

    2016-01-01

    Background/Aim: The BRAFV600E mutation acts as an initiator of cancer development in papillary thyroid carcinoma (PTC). Gene expression changes caused by the BRAFV600E mutation may have an important role in thyroid cancer development. Materials and Methods: To study genomic alterations caused by the BRAFV600E mutation, we made human thyroid cell lines that harbor the wild-type BRAF gene (Nthy/WT) and the V600E mutant-type BRAF gene (Nthy/V600E). Results: Flow cytometry and western blotting showed stable transfection of the BRAF gene. In functional experiments, Nthy/V600E showed increased anchorage-independent growth and invasion through Matrigel, compared to Nthy/WT. Microarray analysis revealed that 2,441 genes were up-regulated in Nthy/V600E compared to Nthy/WT. Gene ontology analysis showed that the up-regulated genes were associated with cell adhesion, migration, and the ERK and MAPK cascade, and pathway analysis showed enrichment in cancer-related pathways. Conclusion: Our Nthy/WT and Nthy/V600E cell line pair could be a suitable model to study the molecular characteristics of BRAFV600E PTC. *These Authors contributed equally to this study. PMID:28031237

  2. Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

    PubMed

    Murray-Stewart, Tracy; Applegren, Nancy B; Devereux, Wendy; Hacker, Amy; Smith, Renee; Wang, Yanlin; Casero, Robert A

    2003-07-15

    Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

  3. Changes in the phenotype of human small cell lung cancer cell lines after transfection and expression of the c-myc proto-oncogene.

    PubMed Central

    Johnson, B E; Battey, J; Linnoila, I; Becker, K L; Makuch, R W; Snider, R H; Carney, D N; Minna, J D

    1986-01-01

    Small cell lung cancer growing in cell culture possesses biologic properties that allow classification into two categories: classic and variant. Compared with classic small cell lung cancer cell lines, variant lines have altered large cell morphology, shorter doubling times, higher cloning efficiencies in soft agarose, and very low levels of L dopa decarboxylase production and bombesin-like immunoreactivity. C-myc is amplified and expressed in some small cell lung cancer cell lines and all c-myc amplified lines studied to date display the variant phenotype. To investigate if c-myc amplification and expression is responsible for the variant phenotype, a normal human c-myc gene was transfected into a cloned classic small cell lung cancer cell line not amplified for or expressing detectable c-myc messenger RNA (mRNA). Clones were isolated with one to six copies of c-myc stably integrated into DNA that expressed c-myc mRNA. In addition, one clone with an integrated neo gene but a deleted c-myc gene was isolated and in this case c-myc was not expressed. C-myc expression in transfected clones was associated with altered large cell morphology, a shorter doubling time, and increased cloning efficiency, but no difference in L dopa decarboxylase levels and bombesin-like immunoreactivity. We conclude increased c-myc expression observed here in transfected clones correlates with some of the phenotypic properties distinguishing c-myc amplified variants from unamplified classic small cell lung cancer lines. Images PMID:3016030

  4. Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin.

    PubMed

    Hu, Li-Zhong; Wan, Ning; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-03-24

    Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

  5. Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin

    NASA Astrophysics Data System (ADS)

    Hu, Li-Zhong; Wan, Ning; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-03-01

    Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

  6. Towards optical cell transfection inside a micro flow cell

    NASA Astrophysics Data System (ADS)

    Breunig, H. G.; Uchugonova, A.; König, K.

    2014-03-01

    For optical transfection, cells are shortly subjected to intense, focused laser radiation which leads to a temporary opening in the cell membrane. Although the method is very efficient and ensures high cell viability, the targeting of single cells with laser pulses is a tedious and slow approach. We present first measurements aiming at an experimental setup which is suitable for high throughput and automated optical cell transfection. In our setup, cells flow through a micro flow cell where they are spatially confined. The laser radiation is focused into the cell in a way that an elongated focal region is realized. This makes the time consuming aiming of the laser beam at individual cells unnecessary and opens the possibility to develop a completely automated system. The elongated laser focal region is realized by a quasi-Bessel beam which is generated by an axicon lens setup and continuously scanned from side to side of the cell. We present test measurements of the newly employed setup and discuss its suitability to be fully integrated into a flow cell sequencing system.

  7. Multiple C4/Slp genes distinguished by expression after transfection.

    PubMed Central

    Robins, D M; Malissen, M; Hood, L; Ferreira, A; Walthall, D; Mitchell, M

    1986-01-01

    The S region of the murine major histocompatibility complex contains two closely related genes: C4, encoding the fourth component of complement, and Slp, encoding sex-limited protein. We cloned these genes from a cosmid library of the B10.W7R strain that does not show androgen regulation of the Slp protein. Restriction site polymorphisms revealed at least four C4-like genes within the Sw7 locus, indicating evolutionary amplification of this region. Transfection of these genes into L cells resulted in expression, processing, and secretion of immunologically correct C4 and Slp proteins. At least two different Slp genes and one C4 gene were capable, after transfection, of expressing C4 and Slp indistinguishable from macrophage-derived protein. A third Slp gene exists within this locus whose recombinant cognate did not express in L cells. Thus, the B10.W7R S region includes one C4 gene and at least three Slp-like genes. Images PMID:3023818

  8. Transfection-mediated recombination of influenza A virus.

    PubMed Central

    Bergmann, M; García-Sastre, A; Palese, P

    1992-01-01

    Several mechanisms, including a high mutation rate and reassortment of genes, have been found to be responsible for the variability of influenza A viruses. RNA recombination would be another mechanism leading to genetic variation; however, recombination has only rarely been reported to occur in influenza viruses. During ribonucleoprotein transfection experiments designed to generate viable influenza viruses from in vitro-synthesized RNA, we discovered several viruses which must have originated from recombination events. The ribonucleoprotein transfection system may enhance the formation of viruses which result from jumping of the viral polymerase between RNAs or from ligation of different viral RNAs. Five different recombinant viruses are described. Two of these, REC1 and REC2, contain a neuraminidase (NA) gene whose defective polyadenylation signal has been repaired via intergenic recombination; 124 and 95 nucleotides have been added, respectively. Another virus, REC5, must have originated by multiple recombination events since it contains a mosaic gene with sequences derived from the NA gene of influenza A/WSN/33 virus and the matrix, polymerase protein PB1, and NA genes of influenza A/PR/8/34 virus. Images PMID:1279208

  9. A label-free bioluminescent sensor for real-time monitoring polynucleotide kinase activity.

    PubMed

    Du, Jiao; Xu, Qinfeng; Lu, Xiaoquan; Zhang, Chun-yang

    2014-08-19

    Polynucleotide kinase (PNK) plays a crucial role in maintaining the genomic stability of cells and is becoming a potential target in the radio-therapeutic treatment of cancers. The fluorescent method is usually used to measure the PNK activity, but it is impossible to obtain the real-time monitoring without the employment of the labeled DNA probes. Here, we report a label-free bioluminescent sensor for PNK activity assay through real-time monitoring of the phosphorylation-dependent DNA ligation reaction. In this bioluminescent sensor, two hairpin DNA probes with 5'-protruding terminal are designed as the phosphate acceptor, and the widely used phosphate donor of ATP is substituted by dCTP. In the absence of PNK, the ligation reaction cannot be triggered due to the lack of 5'-phosphoryl groups in the probes, and the background signal is negligible. With the addition of PNK, the phosphorylation-ligation reaction of the probes is initiated with the release of AMP, and the subsequent conversion of AMP to ATP leads to the generation of distinct bioluminescence signal. The PNK activity assay can be performed in real time by continuously monitoring the bioluminescence signal. This bioluminescent sensor is much simpler, label-free, cost-effective, and free from the autofluorescence interference of biological matrix, and can be further used for quantitative, kinetic, and inhibition assay.

  10. High throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence

    PubMed Central

    Shultzaberger, Ryan K.; Paddock, Mark L.; Katsuki, Takeo; Greenspan, Ralph J.; Golden, Susan S.

    2016-01-01

    The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise, and generate meaningful quantitative measurements of clock output levels for advanced analysis. PMID:25662451

  11. High-throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence.

    PubMed

    Shultzaberger, Ryan K; Paddock, Mark L; Katsuki, Takeo; Greenspan, Ralph J; Golden, Susan S

    2015-01-01

    The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise and generate meaningful quantitative measurements of clock output levels for advanced analysis.

  12. Bioluminescent bioreporter assays for targeted detection of chemical and biological agents

    NASA Astrophysics Data System (ADS)

    Ripp, Steven; Jegier, Pat; Johnson, Courtney; Moser, Scott; Islam, Syed; Sayler, Gary

    2008-04-01

    Bioluminescent bioreporters carrying the bacterial lux gene cassette have been well established for the sensing and monitoring of select chemical agents. Their ability to generate target specific visible light signals with no requirement for extraneous additions of substrate or other hands-on manipulations affords a real-time, repetitive assaying technique that is remarkable in its simplicity and accuracy. Although the predominant application of lux-based bioluminescent bioreporters has been towards chemical compound detection, novel genetic engineering schemes are yielding a variety of new bioreporter systems that extend the lux sensing mechanism beyond mere analyte discrimination. For example, the unique specificity of bacteriophage (bacterial viruses) has been exploited in lux bioluminescent assays for specific identification of foodborne bacterial pathogens such as Escherichia coli O157:H7. With the concurrent ability to interface bioluminescent bioreporter assays onto integrated circuit microluminometers (BBICs; bioluminescent bioreporter integrated circuits), the potential exists for the development of sentinel microchips that can function as environmental monitors for multiplexed recognition of chemical and biological agents in air, food, and water. The size and portability of BBIC biosensors may ultimately provide a deployable, interactive network sensing technology adaptable towards chem/bio defense.

  13. PCR-based detection of bioluminescent microbial populations in Tyrrhenian Sea

    NASA Astrophysics Data System (ADS)

    Gentile, Gabriela; De Luca, Massimo; Denaro, Renata; La Cono, Violetta; Smedile, Francesco; Scarfì, Simona; De Domenico, Emilio; De Domenico, Maria; Yakimov, Michail M.

    2009-05-01

    The present study is focused on the development of a cultivation-independent molecular approach for specific detection of bioluminescent bacteria within microbial communities by direct amplification of luxA gene from environmental DNA. A new set of primers, specifically targeting free-living bioluminescent bacteria, was designed on the base of l uxA sequences available from the public database. Meso- and bathypelagic seawater samples were collected from two stations in Tyrrhenian Sea at the depths of 500 and 2750 m. The same seawater samples also were used to isolate bioluminescent bacteria that were further subjected to luxA and 16S rRNA gene sequencing. PCR products obtained by amplification with designed primers were cloned, and the phylogenetic affiliation of 40 clones was determined. All of them were clustered into three groups, only distantly related to the Photobacterium phosphoreum and Photobacterium kishitanii clades. The half of all clones formed a tight monophyletic clade, while the rest of clones were organized in "compartment"-specific, meso- and bathypelagic ecotypes. No matches with luxA gene sequences of four bioluminescent strains, isolated from the same seawater samples, were observed. These findings indicate that the PCR-based approach developed in present manuscript, allowed us to detect the novel, "yet to be cultivated" lineages of bioluminescent bacteria, which are likely specific for distinct warm bathypelagic realms of Mediterranean Sea.

  14. Development of bioluminescent bioreporters for in vitro and in vivo tracking of Yersinia pestis.

    PubMed

    Sun, Yanwen; Connor, Michael G; Pennington, Jarrod M; Lawrenz, Matthew B

    2012-01-01

    Yersinia pestis causes an acute infection known as the plague. Conventional techniques to enumerate Y. pestis can be labor intensive and do not lend themselves to high throughput assays. In contrast, bioluminescent bioreporters produce light that can be detected using plate readers or optical imaging platforms to monitor bacterial populations as a function of luminescence. Here, we describe the development of two Y. pestis chromosomal-based luxCDABE bioreporters, Lux(PtolC) and Lux(PcysZK). These bioreporters use constitutive promoters to drive expression of luxCDABE that allow for sensitive detection of bacteria via bioluminescence in vitro. Importantly, both bioreporters demonstrate a direct correlation between bacterial numbers and bioluminescence, which allows for bioluminescence to be used to compare bacterial numbers. We demonstrate the use of these bioreporters to test antimicrobial inhibitors (Lux(PtolC)) and monitor intracellular survival (Lux(PtolC) and Lux(PcysZK)) in vitro. Furthermore, we show that Y. pestis infection of the mouse model can be monitored using whole animal optical imaging in real time. Using optical imaging, we observed Y. pestis dissemination and differentiated between virulence phenotypes in live animals via bioluminescence. Finally, we demonstrate that whole animal optical imaging can identify unexpected colonization patterns in mutant-infected animals.

  15. Continuous, real-time bioimaging of chemical bioavailability and toxicology using autonomously bioluminescent human cell lines

    PubMed Central

    Xu, Tingting; Close, Dan M.; Webb, James D.; Price, Sarah L.; Ripp, Steven A.; Sayler, Gary S.

    2015-01-01

    Bioluminescent imaging is an emerging biomedical surveillance strategy that uses external cameras to detect in vivo light generated in small animal models of human physiology or in vitro light generated in tissue culture or tissue scaffold mimics of human anatomy. The most widely utilized of reporters is the firefly luciferase (luc) gene; however, it generates light only upon addition of a chemical substrate, thus only generating intermittent single time point data snapshots. To overcome this disadvantage, we have demonstrated substrate-independent bioluminescent imaging using an optimized bacterial bioluminescence (lux) system. The lux reporter produces bioluminescence autonomously using components found naturally within the cell, thereby allowing imaging to occur continuously and in real-time over the lifetime of the host. We have validated this technology in human cells with demonstrated chemical toxicological profiling against exotoxin exposures at signal strengths comparable to existing luc systems (~1.33 × 107 photons/second). As a proof-in-principle demonstration, we have engineered breast carcinoma cells to express bioluminescence for real-time screening of endocrine disrupting chemicals and validated detection of 17β-estradiol (EC50 = ~ 10 pM). These and other applications of this new reporter technology will be discussed as potential new pathways towards improved models of target chemical bioavailability, toxicology, efficacy, and human safety. PMID:26516295

  16. Continuous, real-time bioimaging of chemical bioavailability and toxicology using autonomously bioluminescent human cell lines

    NASA Astrophysics Data System (ADS)

    Xu, Tingting; Close, Dan M.; Webb, James D.; Price, Sarah L.; Ripp, Steven A.; Sayler, Gary S.

    2013-05-01

    Bioluminescent imaging is an emerging biomedical surveillance strategy that uses external cameras to detect in vivo light generated in small animal models of human physiology or in vitro light generated in tissue culture or tissue scaffold mimics of human anatomy. The most widely utilized of reporters is the firefly luciferase (luc) gene; however, it generates light only upon addition of a chemical substrate, thus only generating intermittent single time point data snapshots. To overcome this disadvantage, we have demonstrated substrate-independent bioluminescent imaging using an optimized bacterial bioluminescence (lux) system. The lux reporter produces bioluminescence autonomously using components found naturally within the cell, thereby allowing imaging to occur continuously and in real-time over the lifetime of the host. We have validated this technology in human cells with demonstrated chemical toxicological profiling against exotoxin exposures at signal strengths comparable to existing luc systems (~1.33 × 107 photons/second). As a proof-in-principle demonstration, we have engineered breast carcinoma cells to express bioluminescence for real-time screening of endocrine disrupting chemicals and validated detection of 17β-estradiol (EC50 = ~ 10 pM). These and other applications of this new reporter technology will be discussed as potential new pathways towards improved models of target chemical bioavailability, toxicology, efficacy, and human safety.

  17. Development of a novel, bioluminescence-based, fungal bioassay for toxicity testing.

    PubMed

    Weitz, Hedda J; Campbell, Colin D; Killham, Ken

    2002-07-01

    Naturally bioluminescent fungi, Armillaria mellea and Mycena citricolor, were used to develop a novel, bioluminescence-based bioassay for toxicity testing. Bioassays were carried out to assess the toxicity of 3,5-dichlorophenol (3,5-DCP), pentachlorophenol (PCP), copper and zinc. The results suggested that 60 min was a suitable exposure time for the bioassay. Light reduction was observed in response to 3,5-DCP, PCP and Cu for both A. mellea and M. citricolor, but to Zn only for A. mellea. Armillaria mellea was significantly less sensitive to 3,5-DCP and PCP than M. citricolor. The EC50 values for A. mellea and M. citricolor were similar to EC50 values for 3,5-DCP, PCP and Cu (but not Zn) of bioluminescence-based bacterial biosensors. They were also similar to EC50 values for Cu and Zn of a bioluminescence-based yeast biosensor. The results highlighted the importance of using both prokaryotic and eukaryotic biosensors. The novel bioassay provides a rapid and sensitive method to assess bioavailability of pollutants as well as a method to determine their toxicity to filamentous fungi. It also expands the range of organisms that can be used for bioluminescence-based toxicity testing by complementing existing biosensors.

  18. Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

    PubMed Central

    Close, Dan M.; Patterson, Stacey S.; Ripp, Steven; Baek, Seung J.; Sanseverino, John; Sayler, Gary S.

    2010-01-01

    Background The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo. Methodology/Principal Findings Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH2) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background. Conclusions/Significance The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies. PMID:20805991

  19. A posteriori correction for source decay in 3D bioluminescent source localization using multiview measured data

    NASA Astrophysics Data System (ADS)

    Sun, Li; Wang, Pu; Tian, Jie; Liu, Dan; Wang, Ruifang

    2009-02-01

    As a novel optical molecular imaging technique, bioluminescence tomography (BLT) can be used to monitor the biological activities non-invasively at the cellular and molecular levels. In most of known BLT studies, however, the time variation of the bioluminescent source is neglected. It gives rise to the inconsistent views during the multiview continuous wave measurement. In other words, the real measured data from different measured views come from 'different' bioluminescent sources. It could bring large errors in bioluminescence reconstruction. In this paper, a posteriori correction strategy for adaptive FEM-based reconstruction is proposed and developed. The method helps to improve the source localization considering the bioluminescent energy variance during the multiview measurement. In the method, the correction for boundary signals by means of a posteriori correction strategy, which adopts the energy ratio of measured data in the overlapping domains between the adjacent measurements as the correcting factor, can eliminate the effect of the inconsistent views. Then the adaptive mesh refinement with a posteriori error estimation helps to improve the precision and efficiency of BLT reconstruction. In addition, a priori permissible source region selection based on the surface measured data further reduces the ill-posedness of BLT and enhances numerical stability. Finally, three-dimension numerical simulations using the heterogeneous phantom are performed. The numerically measured data is generated by Monte Carlo (MC) method which is known as the Gold standard and can avoid the inverse crime. The reconstructed result with correction shows more accuracy compared to that without correction.

  20. Environmental and synthetic sulphydryl group inhibitors: effects on bioluminescence and respiration in Vibrio fischeri.

    PubMed

    Kalciene, Virginija; Cetkauskaite, Anolda

    2007-03-01

    Elemental sulphur (as S0 and S8) is abundant in anaerobic sediments and soil, and is highly toxic in the Vibrio fischeri bioluminescence test. This mode of S0 action remains uncertain. The objective of this research was the analysis of the toxic effects of S0 on bioluminescence and respiration in V. fischeri, in joint action with N-ethylmaleimide (NEM) or 2,4-dithio-DL-threitol (DTT), which are -SH group inhibiting and maintaining synthetic agents, respectively. Non-toxic DTT immediately protected cell bioluminescence against S0 inhibition at low (5.5ppb) and high (55ppb) concentrations of S0, whilst restoration of the inhibitory effect of S0 took up to 30 minutes. NEM (62.5ppb) diminished cell bioluminescence by up to 50% after 5 minutes, but after 60 minutes, the inhibition reached 100%. DTT restored the bioluminescence function inhibited in vivo and in vitro by S0 and NEM. Enhancement of cell respiration by up to 20% and 33% was observed at 2.2ppm of S0 and 36.8ppm of 2,4-dinitrophenol (2,4-DNP; an uncoupler of oxidative phosphorylation), respectively; whilst NEM (3.1ppm) caused a reduction of up to 40%. This comparative analysis confirmed that S0 has multiple modes of action--it acts as both an -SH group inhibitor and an uncoupler of oxidative phosphorylation in V. fischeri cells.

  1. In vitro influence of hypoxia on bioluminescence imaging in brain tumor cells

    NASA Astrophysics Data System (ADS)

    Moriyama, Eduardo H.; Jarvi, Mark; Niedre, Mark; Mocanu, Joseph D.; Moriyama, Yumi; Li, Buhong; Lilge, Lothar; Wilson, Brian C.

    2007-02-01

    Bioluminescence Imaging (BLI) has been employed as an imaging modality to identify and characterize fundamental processes related to cancer development and response at cellular and molecular levels. This technique is based on the reaction of luciferin with oxygen in the presence of luciferase and ATP. A major concern in this technique is that tumors are generally hypoxic, either constitutively and/or as a result of treatment, therefore the oxygen available for the bioluminescence reaction could possibly be reduced to limiting levels, and thus leading to underestimation of the actual number of luciferase-labeled cells during in vivo procedures. In this report, we present the initial in vitro results of the oxygen dependence of the bioluminescence signal in rat gliosarcoma 9L cells tagged with the luciferase gene (9L luc cells). Bioluminescence photon emission from cells exposed to different oxygen tensions was detected by a sensitive CCD camera upon exposure to luciferin. The results showed that bioluminescence signal decreased at administered pO II levels below about 5%, falling by approximately 50% at 0.2% pO II. Additional experiments showed that changes in BLI was due to the cell inability to maintain normal levels of ATP during the hypoxic period reducing the ATP concentration to limiting levels for BLI.

  2. Spectrally resolved bioluminescence tomography with the third-order simplified spherical harmonics approximation

    NASA Astrophysics Data System (ADS)

    Lu, Yujie; Douraghy, Ali; Machado, Hidevaldo B.; Stout, David; Tian, Jie; Herschman, Harvey; Chatziioannou, Arion F.

    2009-11-01

    Bioluminescence imaging has been extensively applied to in vivo small animal imaging. Quantitative three-dimensional bioluminescent source information obtained by using bioluminescence tomography can directly and much more accurately reflect biological changes as opposed to planar bioluminescence imaging. Preliminary simulated and experimental reconstruction results demonstrate the feasibility and promise of bioluminescence tomography. However, the use of multiple approximations, particularly the diffusion approximation theory, affects the quality of in vivo small animal-based image reconstructions. In the development of new reconstruction algorithms, high-order approximation models of the radiative transfer equation and spectrally resolved data introduce new challenges to the reconstruction algorithm and speed. In this paper, an SP3-based (the third-order simplified spherical harmonics approximation) spectrally resolved reconstruction algorithm is proposed. The simple linear relationship between the unknown source distribution and the spectrally resolved data is established in this algorithm. A parallel version of this algorithm is realized, making BLT reconstruction feasible for the whole body of small animals especially for fine spatial domain discretization. In simulation validations, the proposed algorithm shows improved reconstruction quality compared with diffusion approximation-based methods when high absorption, superficial sources and detection modes are considered. In addition, comparisons between fine and coarse mesh-based BLT reconstructions show the effects of numerical errors in reconstruction image quality. Finally, BLT reconstructions using in vivo mouse experiments further demonstrate the potential and effectiveness of the SP3-based reconstruction algorithm.

  3. Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

    PubMed Central

    2011-01-01

    Background Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. Results The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet

  4. Tomographic bioluminescence imaging by an iteratively re-weighted minimization

    NASA Astrophysics Data System (ADS)

    Wu, Ping; Liu, Kai; Xue, Zhenwen; Guo, Wei; Qin, Chenghu; Tian, Jie

    2012-03-01

    Tomographic bioluminescence imaging (TBI), with visible light emission in living organisms, is an effective way of molecular imaging, which allows for the study of ongoing tumor biological processes in vivo and non-invasively. This newly developed technology enables three-dimensional accuracy localization and quantitative analysis of the target tumor cells in small animal via reconstructing the images acquired by the high-resolution imaging system. Due to the difficulty of reconstruction, which is often referred to an ill-posed inverse problem, continuous efforts are still made to find more practical and efficient approaches. In this paper, an iteratively re-weighted minimization (IRM) has been applied to reconstruct the entire source distribution, which is known as sparse signals, inside the target tissue with the limited outgoing photon density on its boundary. By introducing a weight function into the objective function, we convert the lp norm problem into a more simple form of l2 norm to reduce the computational complexity. The weight function is updated in each iterative step to compute the final optimal solution more efficiently. This method is proved to be robust to different parameters, and mouse experiments are conducted to validate the feasibility of IRM approach, which is also reliable at whole-body imaging.

  5. Detection of a bioluminescent milky sea from space

    PubMed Central

    Miller, Steven D.; Haddock, Steven H. D.; Elvidge, Christopher D.; Lee, Thomas F.

    2005-01-01

    On many occasions over the centuries, mariners have reported witnessing surreal nocturnal displays where the surface of the sea produces an intense, uniform, and sustained glow that extends to the horizon in all directions. Although such emissions cannot be fully reconciled with the known features of any light-emitting organism, these so-called “milky seas” are hypothesized to be manifestations of unusually strong bioluminescence produced by colonies of bacteria in association with a microalgal bloom in the surface waters. Because of their ephemeral nature and the paucity of scientific observations, an explanation of milky seas has remained elusive. Here, we report the first satellite observations of the phenomenon. An ≈15,400-km2 area of the northwestern Indian Ocean, roughly the size of the state of Connecticut, was observed to glow over 3 consecutive nights, corroborated on the first night by a ship-based account. This unanticipated application of satellite remote-sensing technology provides insights pertaining to the formation and scale of these poorly understood events. PMID:16186481

  6. Detection of a bioluminescent milky sea from space.

    PubMed

    Miller, Steven D; Haddock, Steven H D; Elvidge, Christopher D; Lee, Thomas F

    2005-10-04

    On many occasions over the centuries, mariners have reported witnessing surreal nocturnal displays where the surface of the sea produces an intense, uniform, and sustained glow that extends to the horizon in all directions. Although such emissions cannot be fully reconciled with the known features of any light-emitting organism, these so-called "milky seas" are hypothesized to be manifestations of unusually strong bioluminescence produced by colonies of bacteria in association with a microalgal bloom in the surface waters. Because of their ephemeral nature and the paucity of scientific observations, an explanation of milky seas has remained elusive. Here, we report the first satellite observations of the phenomenon. An approximately 15,400-km(2) area of the northwestern Indian Ocean, roughly the size of the state of Connecticut, was observed to glow over 3 consecutive nights, corroborated on the first night by a ship-based account. This unanticipated application of satellite remote-sensing technology provides insights pertaining to the formation and scale of these poorly understood events.

  7. Detection of Metal and Organometallic Compounds with Bioluminescent Bacterial Bioassays.

    PubMed

    Durand, M J; Hua, A; Jouanneau, S; Cregut, M; Thouand, G

    2015-10-17

    Chemical detection of metal and organometallic compounds is very specific and sensitive, but these techniques are time consuming and expensive. Although these techniques provide information about the concentrations of compounds, they fail to inform us about the toxicity of a sample. Because the toxic effects of metals and organometallic compounds are influenced by a multitude of environmental factors, such as pH, the presence of chelating agents, speciation, and organic matter, bioassays have been developed for ecotoxicological studies. Among these bioassays, recombinant luminescent bacteria have been developed over the past 20 years, and many of them are specific for the detection of metals and metalloids. These bioassays are simple to use, are inexpensive, and provide information on the bioavailable fraction of metals and organometals. Thus, they are an essential complementary tool for providing information beyond chemical analysis. In this chapter, we propose to investigate the detection of metals and organometallic compounds with bioluminescent bacterial bioassays and the applications of these bioassays to environmental samples. Graphical Abstract.

  8. Bioluminescence to reveal structure and interaction of coastal planktonic communities

    NASA Astrophysics Data System (ADS)

    Moline, Mark A.; Blackwell, Shelley M.; Case, James F.; Haddock, Steven H. D.; Herren, Christen M.; Orrico, Cristina M.; Terrill, Eric

    2009-02-01

    Ecosystem function will in large part be determined by functional groups present in biological communities. The simplest distinction with respect to functional groups of an ecosystem is the differentiation between primary and secondary producers. A challenge thus far has been to examine these groups simultaneously with sufficient temporal and spatial resolution for observations to be relevant to the scales of change in coastal oceans. This study takes advantage of general differences in the bioluminescence flash kinetics between planktonic dinoflagellates and zooplankton to measure relative abundances of the two groups within the same-time space volume. This novel approach for distinguishing these general classifications using a single sensor is validated using fluorescence data and exclusion experiments. The approach is then applied to data collected from an autonomous underwater vehicle surveying >500 km in Monterey Bay and San Luis Obispo Bay, CA during the summers of 2002-2004. The approach also reveals that identifying trophic interaction between the two planktonic communities may also be possible.

  9. PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells

    PubMed Central

    Inada, Emi; Saitoh, Issei; Watanabe, Satoshi; Aoki, Reiji; Miura, Hiromi; Ohtsuka, Masato; Murakami, Tomoya; Sawami, Tadashi; Yamasaki, Youichi; Sato, Masahiro

    2015-01-01

    The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine. PMID:26208039

  10. Functional expression of recombinant human ribonuclease/angiogenin inhibitor in stably transformed Drosophila melanogaster S2 cells.

    PubMed

    Park, Jong-Hwa; Hwang, In-Sook; Kim, Kyung-Il; Lee, Jong-Min; Park, Young-Min; Park, Chang-Ho; Chung, In Sik

    2008-05-01

    A recombinant plasmid harboring heterologous genes coding human ribonuclease/angiogenin inhibitor (RAI) was expressed in stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing RAI were isolated after 4 weeks of selection with hygromycin B. Recombinant RAI with a molecular weight of 50 kDa was detected in the intracellular (cell) and extracellular (medium) fractions of S2 cells. Recombinant RAI was purified from the extracellular fraction using a two-step purification scheme comprised of Ni-NTA and ion-exchange chromatography. Purified RAI migrated on SDS-PAGE as a single band in the elution fraction containing 300 mM NaCl. The ribonuclease inhibitor activity of purified RAI was measured using yeast tRNA and RNase A. Purified RAI exhibited an activity of approximately 8 U mug(-1) for the inhibition of RNA degradation by RNase A. Cultivation of stably transformed S2 cells using HyQ((R))SFX-insect MP medium increased cell growth by 79% and approximately doubled the production of recombinant RAI.

  11. Protease-sensitive transfection of Bacillus subtilis with bacteriophage GA-1 DNA: a probable case of heterologous transfection.

    PubMed

    Arwert, F; Venema, G

    1974-03-01

    The host bacterium of bacteriophage GA-1, Bacillus sp. G1R, was compared with respect to its taxonomic relationship to Bacillus subtilis, B. licheniformis, and B. pumilis. The physiological-biochemical properties of Bacillus sp. G1R are equal to those of B. licheniformis, but the thermal denaturation midpoint of G1R DNA differs by 3 C and the buoyant density by 0.005 g/cm(3) from that of B. licheniformis. Transformation with G1R donor DNA was neither observed in B. licheniformis nor in B. subtilis-competent recipients. Bacteriophage GA-1 shows neither infectivity on B. licheniformis nor on B. subtilis. However, infection of competent B. subtilis cultures with phenol-extracted GA-1 DNA results in the production of infective GA-1 particles. The transfecting activity of GA-1 DNA is destroyed by treatment with proteolytic enzymes. Resistance of transfecting DNA to inactivation by trypsin develops earlier than that to inactivation by DNase. Protease-treated GA-1 DNA competes with transforming DNA to approximately the same extent as does untreated GA-1 DNA, suggesting that uptake of GA-1 DNA is not affected by protease treatment. CsCl density gradient centrifugation reveals that the density of trypsinized GA-1 DNA is 0.004 g/cm(3) greater than that of untreated DNA.

  12. Synthetic strategies for controlling inter- and intramolecular interactions: Applications in single-molecule fluorescence imaging, bioluminescence imaging, and palladium catalysis

    NASA Astrophysics Data System (ADS)

    Conley, Nicholas R.

    proximity of the Cy3 and Cy5 fluorophores, behaves as an optical photoswitch in the presence of a thiol reagent. This unique property was employed to achieve sub-diffraction-limited imaging of the stalks of Caulobacter crescentus cells with 30-nm resolution using STORM (stochastic optical reconstruction microscopy). Lastly, the synthesis of the first selenium analogue of firefly luciferin is described, and this analogue is shown to be a competent substrate for firefly luciferase (fLuc). Remarkably, it exhibits red-shifted bioluminescence emission relative to the native sulfur analogue. The in vivo performance of the selenium and sulfur analogues in imaging are compared by tail-vein injection into nude mice bearing subcutaneous tumor xenografts of a human breast cancer cell line that was stably transduced to express fLuc. Part II of this thesis begins by addressing design considerations in the development of palladium catalysts that effect oxidative transformations under mild conditions (i.e., 1 atm air, room temperature) using molecular oxygen as the terminal oxidant. A newly synthesized cationic palladium complex, [(2,9-dimethylphenanthroline)Pd(OAc)]2[OTf]2, is shown to catalyze aerobic alcohol oxidation under such conditions with an unprecedented initial turnover frequency, but the presence of partially reduced oxygen species results in competitive ligand oxidation with concomitant decrease in catalyst activity. To remedy this, oxidatively resistant ligands, which are essential for the development of next-generation, high-turnover-frequency palladium catalysts that utilize oxygen as a terminal oxidant, have been prepared and effectively employed. In addition, the first general palladium-catalyzed route to the carbonylation of diols is reported. In this system, carbon monoxide (1 atm) serves the carbonyl source, (2,9-dimethylphenanthroline)Pd(OAc) 2 acts as the catalyst, and N-chlorosuccinimide and iodosobenzene are the oxidants for 1,2- and 1,3-diols, respectively. This

  13. Mouse in utero electroporation: controlled spatiotemporal gene transfection.

    PubMed

    Matsui, Asuka; Yoshida, Aya C; Kubota, Mayumi; Ogawa, Masaharu; Shimogori, Tomomi

    2011-08-15

    In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early

  14. Iterative method for bioluminescence tomography based on the radiative transport equation

    NASA Astrophysics Data System (ADS)

    Cong, Wenxiang; Wang, Ge

    2006-08-01

    Bioluminescence tomography (BLT) is a new molecular imaging modality, which helps study cancer and other diseases, develop drugs, and so on. This technology localizes and quantifies a bioluminescent source inside a living transgenic mouse, and is very useful in many biomedical applications. In this paper, we propose a novel algorithm based on the radiative transport equation to reconstruct the bioluminescence source distribution from data measured on the external surface of a mouse. Our approach transforms the transport equation into an integral equation of the second kind, and establishes a linear system to link the measured photon fluence rate with the unknown light source variables. A regularization measure is taken to overcome the ill-posedness of the inverse problem. Then, an iterative optimization technique with a simple constrain is employed to compute the desirable solution. The physical phantom experiments have been performed to demonstrate the feasibility of the reconstruction method, and evaluate its performance in terms of source location and power estimation.

  15. Application of ATP bioluminescence for evaluation of surface cleanliness of milking equipment.

    PubMed

    Vilar, M J; Rodríguez-Otero, J L; Diéguez, F J; Sanjuán, M L; Yus, E

    2008-07-31

    The ATP bioluminescence method was used to evaluate the cleanliness of milking equipment surfaces (teat cup rubbers, teat dip containers, milk receivers, and pipeline joints) in dairy farms in Galicia (northwest Spain) with parlour, pipeline tie-stall or bucket tie-stall milking systems. The cleanest surfaces were teat cup rubbers. The use of non-chlorinated water for cleaning, and of pipeline or bucket tie-stall milking systems, was associated with high ATP bioluminescence values. However, ATP bioluminescence values only explained 12% of the variability in bulk-tank bacterial count; this is attributable to the importance of other factors (notably the correct functioning of the tank cooling system) for maintenance of low bacterial count.

  16. Characterization of an anthraquinone fluor from the bioluminescent, pelagic polychaete Tomopteris

    PubMed Central

    Francis, Warren R; Powers, Meghan L; Haddock, Steven H D

    2014-01-01

    Tomopteris is a cosmopolitan genus of polychaetes. Many species produce yellow luminescence in the parapodia when stimulated. Yellow bioluminescence is rare in the ocean, and the components of this luminescent reaction have not been identified. Only a brief description, half a century ago, noted fluorescence in the parapodia with a remarkably similar spectrum to the bioluminescence, which suggested that it may be the luciferin or terminal light-emitter. Here, we report the isolation of the fluorescent yellow–orange pigment found in the luminous exudate and in the body of the animals. Liquid chromatography-mass spectrometry revealed the mass to be 270 m/z with a molecular formula of C15H10O5, which ultimately was shown to be aloe-emodin, an anthraquinone previously found in plants. We speculate that aloe-emodin could be a factor for resonant-energy transfer or the oxyluciferin for Tomopteris bioluminescence. PMID:24760626

  17. Characterization of an anthraquinone fluor from the bioluminescent, pelagic polychaete Tomopteris.

    PubMed

    Francis, Warren R; Powers, Meghan L; Haddock, Steven H D

    2014-12-01

    Tomopteris is a cosmopolitan genus of polychaetes. Many species produce yellow luminescence in the parapodia when stimulated. Yellow bioluminescence is rare in the ocean, and the components of this luminescent reaction have not been identified. Only a brief description, half a century ago, noted fluorescence in the parapodia with a remarkably similar spectrum to the bioluminescence, which suggested that it may be the luciferin or terminal light-emitter. Here, we report the isolation of the fluorescent yellow-orange pigment found in the luminous exudate and in the body of the animals. Liquid chromatography-mass spectrometry revealed the mass to be 270 m/z with a molecular formula of C(15)H(10)O(5), which ultimately was shown to be aloe-emodin, an anthraquinone previously found in plants. We speculate that aloe-emodin could be a factor for resonant-energy transfer or the oxyluciferin for Tomopteris bioluminescence.

  18. Four new bioluminescent taxa of Mycena sect. Calodontes from Peninsular Malaysia.

    PubMed

    Chew, Audrey L C; Tan, Yee-Shin; Desjardin, Dennis E; Musa, Md Yusoff; Sabaratnam, Vikineswary

    2014-01-01

    Three new species and one new variety of bioluminescent Mycena collected from Peninsular Malaysia are described herein. All new species belong to Mycena sect. Calodontes in what is known as the Mycena pura complex. Comprehensive descriptions, photographs, illustrations and comparisons with phenetically similar species are provided. Molecular sequences data from the nuclear internal transcribed spacers (ITS-1 and ITS-2, including the 5.8S rRNA) were used to infer relationships within sect. Calodontes. Axenic cultures were obtained to provide data on culture morphology. This is the first published photographic documentation of bioluminescent basidiomes of members of Mycena sect. Calodontes. Also, this addition brings the total known bioluminescent fungi to 77 species.

  19. The stimulation of bioluminescence in Photobacterium leiognathi as a potential prescreen for antitumor agents.

    PubMed

    Steinberg, D A; Peterson, G A; White, R J; Maiese, W M

    1985-10-01

    The stimulation of bioluminescence in Photobacterium leiognathi has previously been described as a test for genotoxic compounds. An adaptation of this procedure has been developed which uses a dim variant of P. leiognathi and permits the prescreening of microbial fermentation broths for potential antitumor agents. Bioluminescence in this organism was stimulated by compounds which bind to DNA or affect DNA synthesis. Antibiotics with target sites such as protein, cell wall or RNA synthesis, did not alter bioluminescence. Fermentation broths from over 5,000 soil isolates were prescreened in this assay and 95 (1.6%) were defined as active. Further analysis of selected cultures suggested that about half produced compound(s) with DNA-binding activity. These results suggest that the photobacterium induction assay (PIA) may be useful as a prescreen for potential antitumor agents. The assay is rapid, simple and requires only microgram quantities of material for testing.

  20. [Bioluminescent analysis of the SOS-response of Escherichia coli cells].

    PubMed

    Ptistsyn, L R

    1996-03-01

    We constructed a recombinant plasmid pPLS-1 to estimate the level of SOS response in Escherichia coli by the bioluminescent method. A 6.7-kb promoterless operon of bioluminescence from Photobacterium leiognathi was cloned into a pBR322 vector, in which its expression was controlled by the SOS promoter of gene cda from a plasmid ColD. The sequence between the 5'-terminal Sph1 site of the operon and start codon ATG of the luxC gene was shown to be 56 bp in length and had no effect on the level of light emission. SOS-inducing potency of six mutagenic substances was tested in E. coli strain C600(pPLS-1). The bioluminescent method proved to be very sensitive for estimating the level of SOS response. The results obtained by this method showed good correlation with results obtained by SOS Chromotest, umu-test, and Ames' test.

  1. A two-hour antibiotic susceptibility test by ATP-bioluminescence.

    PubMed

    March Rosselló, Gabriel Alberto; García-Loygorri Jordán de Urries, María Cristina; Gutiérrez Rodríguez, María Purificación; Simarro Grande, María; Orduña Domingo, Antonio; Bratos Pérez, Miguel Ángel

    2016-01-01

    The antibiotic susceptibility test (AST) in Clinical Microbiology laboratories is still time-consuming, and most procedures take 24h to yield results. In this study, a rapid antimicrobial susceptibility test using ATP-bioluminescence has been developed. The design of method was performed using five ATCC collection strains of known susceptibility. This procedure was then validated against standard commercial methods on 10 strains of enterococci, 10 staphylococci, 10 non-fermenting gram negative bacilli, and 13 Enterobacteriaceae from patients. The agreement obtained in the sensitivity between the ATP-bioluminescence method and commercial methods (E-test, MicroScan and VITEK2) was 100%. In summary, the preliminary results obtained in this work show that the ATP-bioluminescence method could provide a fast and reliable AST in two hours.

  2. 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.

  3. Chemiluminescence and Bioluminescence as an Excitation Source in the Photodynamic Therapy of Cancer: A Critical Review.

    PubMed

    Magalhães, Carla M; Esteves da Silva, Joaquim C G; Pinto da Silva, Luís

    2016-08-04

    Photodynamic therapy (PDT) of cancer is known for its limited number of side effects, and requires light, oxygen and photosensitizer. However, PDT is limited by poor penetration of light into deeply localized tissues, and the use of external light sources is required. Thus, researchers have been studying ways to improve the effectiveness of this phototherapy and expand it for the treatment of the deepest cancers, by using chemiluminescent or bioluminescent formulations to excite the photosensitizer by intracellular generation of light. The aim of this Minireview is to give a précis of the most important general chemi-/bioluminescence mechanisms and to analyze several studies that apply them for PDT. These studies have demonstrated the potential of utilizing chemi-/bioluminescence as excitation source in the PDT of cancer, besides combining new approaches to overcome the limitations of this mode of treatment.

  4. Monitoring of bacterial contamination of dental unit water lines using adenosine triphosphate bioluminescence.

    PubMed

    Watanabe, A; Tamaki, N; Yokota, K; Matsuyama, M; Kokeguchi, S

    2016-12-01

    Bacterial contamination of dental unit waterlines (DUWLs) was evaluated using ATP bioluminescence analysis and a conventional culture method. Water samples (N=44) from DUWLs were investigated for heterotrophic bacteria by culture on R2A agar, which gave counts ranging from 1.4×10(3) to 2.7×10(5) cfu/mL. The ATP bioluminescence results for DUWL samples ranged from 6 to 1189 relative light units and could be obtained within 1min; these correlated well with the culture results (r=0.727-0.855). We conclude that the results of the ATP bioluminescence assay accurately reflect the results of conventional culture-based testing. This method is potentially useful for rapid and simple monitoring of DUWL bacterial contamination.

  5. Regulated bioluminescence as a tool for bioremediation process monitoring and control of bacterial cultures

    NASA Technical Reports Server (NTRS)

    Burlage, Robert S.; Heitzer, Armin; Digrazia, Philip M.

    1991-01-01

    An effective on-line monitoring technique for toxic waste bioremediation using bioluminescent microorganisms has shown great potential for the description and optimization of biological processes. The lux genes of the bacterium Vibrio fischeri are used by this species to produce visible light. The lux genes can be genetically fused to the control region of a catabolic gene, with the result that bioluminescence is produced whenever the catabolic gene is induced. Thus the detection of light from a sample indicates that genetic expression from a specific gene is occurring. This technique was used to monitor biodegradation of specific contaminants from waste sites. For these studies, fusions between the lux genes and the operons for naphthalene and toluene/xylene degradation were constructed. Strains carrying one of these fusions respond sensitively and specifically to target substrates. Bioluminescence from these cultures can be rapidly measured in a nondestructive and noninvasive manner. The potential for this technique in this and other biological systems is discussed.

  6. The use of bioluminescent biotests for study of natural and laboratory aquatic ecosystems.

    PubMed

    Kratasyuk, V A; Esimbekova, E N; Gladyshev, M I; Khromichek, E B; Kuznetsov, A M; Ivanova, E A

    2001-03-01

    A set of bioluminescent tests was developed to monitor water quality in natural and laboratory ecosystems. It consisted of four bioluminescent systems: luminous bacteria, coupled enzyme system NADH:FMN-oxidoreductase-luciferase and triplet enzyme systems with alcohol dehydrogenase and trypsin. The set of biotests was applied for a small forest pond (Siberia, Russia), laboratory microecosystems polluted with benzoquinone and a batch culture of blue-green algae. Thereby effects of natural water compared to those of models of heavy pollution and "bloom" of blue-greens on the bioluminescent tests were revealed. The set of biotests was not affected by a natural seasonal variability of water quality in the unpolluted pond, but responded to the heavy pollution and the "bloom" of blue-greens. The set of biotests could be recommended as the alarm test to control the acute toxicity of natural water bodies.

  7. In vivo imaging of bioluminescent Pseudomonas aeruginosa in an acute murine airway infection model.

    PubMed

    Munder, Antje; Wölbeling, Florian; Klockgether, Jens; Wiehlmann, Lutz; Tümmler, Burkhard

    2014-10-01

    Non-invasive bioluminescence imaging allows the analysis of infectious diseases in small animal models. In this study, an acute airway infection of C3H/HeN mice with luxCDABE transformed Pseudomonas aeruginosa TBCF10839 and an isogenic transposon mutant was followed by optical imaging in vivo. Using the disease-causing dose of 2.0 × 10(6) CFU of the cystic fibrosis airway isolate TBCF10839, subtle luminescence of the lungs was inconsistently visible for the first hour after infection. Conversely, using a 100-fold higher dose of the strongly virulence-attenuated transposon mutant, the robust signal of bioluminescent bacteria increased over 24 h. To monitor murine airway infections with P. aeruginosa in vivo by bioluminescence, one should select an attenuated mutant of a virulent strain or a wild type strain that naturally lacks virulence determinants and/or that has acquired a low virulence persister phenotype by patho-adaptive mutations.

  8. Genetically encoded bioluminescent voltage indicator for multi-purpose use in wide range of bioimaging

    PubMed Central

    Inagaki, Shigenori; Tsutsui, Hidekazu; Suzuki, Kazushi; Agetsuma, Masakazu; Arai, Yoshiyuki; Jinno, Yuka; Bai, Guirong; Daniels, Matthew J.; Okamura, Yasushi; Matsuda, Tomoki; Nagai, Takeharu

    2017-01-01

    We report development of the first genetically encoded bioluminescent indicator for membrane voltage called LOTUS-V. Since it is bioluminescent, imaging LOTUS-V does not require external light illumination. This allows bidirectional optogenetic control of cellular activity triggered by Channelrhodopsin2 and Halorhodopsin during voltage imaging. The other advantage of LOTUS-V is the robustness of a signal-to-background ratio (SBR) wherever it expressed, even in the specimens where autofluorescence from environment severely interferes fluorescence imaging. Through imaging of moving cardiomyocyte aggregates, we demonstrated the advantages of LOTUS-V in long-term imaging are attributable to the absence of phototoxicity, and photobleaching in bioluminescent imaging, combined with the ratiometric aspect of LOTUS-V design. Collectively LOTUS-V extends the scope of excitable cell control and simultaneous voltage phenotyping, which should enable applications in bioscience, medicine and pharmacology previously not possible. PMID:28205521

  9. Use of Bioluminescence Markers To Detect Pseudomonas spp. in the Rhizosphere

    PubMed Central

    de Weger, Letty A.; Dunbar, Paul; Mahafee, Walter F.; Lugtenberg, Ben J. J.; Sayler, Gary S.

    1991-01-01

    The use of bioluminescence as a sensitive marker for detection of Pseudomonas spp. in the rhizosphere was investigated. Continuous expression of the luxCDABE genes, required for bioluminescence, was not detectable in the rhizosphere. However, when either a naphthalene-inducible luxCDABE construct or a constitutive luxAB construct (coding only for the luciferase) was introduced into the Pseudomonas cells, light emission could be initiated just prior to measurement by the addition of naphthalene or the substrate for luciferase, n-decyl aldehyde, respectively. These Pseudomonas cells could successfully be detected in the rhizosphere by using autophotography or optical fiber light measurement techniques. Detection required the presence of 103 to 104 CFU/cm of root, showing that the bioluminescence technique is at least 1,000-fold more sensitive than β-galactosidase-based systems. Images PMID:16348610

  10. Use of bioluminescence markers to detect Pseudomonas spp. in the Rhizosphere

    SciTech Connect

    De Weger, L.A.; Lugtenberg, B.J.J. ); Dunbar, P.; Sayler, G.S. ); Mahafee, W.F. )

    1991-12-01

    The use of bioluminescence as a sensitive marker for detection of Pseudomonas spp. in the rhizosphere was investigated. Continuous expression of the luxCDABE genes, required for bioluminescence, was not detectable in the rhizosphere. However, when either a naphthalene-inducible luxCDABE construct or a constitutive luxAB construct (coding only for the luciferase) was introduced into the Pseudomonas cells, light emission could be initiated just prior to measurement by the addition of naphthalene or the substrate for luciferase, n-decyl aldehyde, respectively. These Pseudomonas cells could successfully be detected in rhizosphere by using autophotography or optical fiber light measurement techniques. Detection required the presence of 10{sup 3} to 10{sup 4} CFU/cm of root, showing that the bioluminescence technique is at least 1,000-fold more sensitive than {beta}-galactosidase-based systems.

  11. Monitoring of recombinant protein production using bioluminescence in a semiautomated fermentation process.

    PubMed

    Trezzani, I; Nadri, M; Dorel, C; Lejeune, P; Bellalou, J; Lieto, J; Hammouri, H; Longin, R; Dhurjati, P

    2003-01-01

    On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes.

  12. Bioluminescent signals spatially amplified by wavelength-specific diffusion through the shell of a marine snail

    PubMed Central

    Deheyn, Dimitri D.; Wilson, Nerida G.

    2011-01-01

    Some living organisms produce visible light (bioluminescence) for intra- or interspecific visual communication. Here, we describe a remarkable bioluminescent adaptation in the marine snail Hinea brasiliana. This species produces a luminous display in response to mechanical stimulation caused by encounters with other motile organisms. The light is produced from discrete areas on the snail's body beneath the snail's shell, and must thus overcome this structural barrier to be viewed by an external receiver. The diffusion and transmission efficiency of the shell is greater than a commercial diffuser reference material. Most strikingly, the shell, although opaque and pigmented, selectively diffuses the blue-green wavelength of the species bioluminescence. This diffusion generates a luminous display that is enlarged relative to the original light source. This unusual shell thus allows spatially amplified outward transmission of light communication signals from the snail, while allowing the animal to remain safely inside its hard protective shell. PMID:21159673

  13. Preconcentration and detection of mercury with bioluminescent bioreporter E. coli ARL1.

    PubMed

    Solovyev, Andrey I; Koštejn, Martin; Kuncova, Gabriela; Dostálek, Pavel; Rohovec, Jan; Navrátil, Tomáš

    2015-10-01

    Cell wall envelopes treated with sodium hydroxide and spray-dried were used as mercury sorbents. The sorbent having sorption capacity 17.7 ± 0.1 μmol/g determined was employed for preconcentration of mercury containing 1-10 ng/L. After preconcentration, bioavailable mercury was detected in samples of soil, stream, and tap water via induction of bioluminescence of E. coli ARL1. Iron and manganese at concentrations of tenth microgram per liter interfered bioluminescence detection of mercury. In tap water was detected semiquantitatively 0.127 ± 0.1 nmol/L by the induction of bioluminescence of E. coli ARL1 in medium with tryptone after preconcentration using a method of standard addition.

  14. Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay.

    PubMed

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-08-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87(N). Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.

  15. Enhancing magnetic nanoparticle-based DNA transfection: Intracellular-active cassette features

    NASA Astrophysics Data System (ADS)

    Vernon, Matthew Martin

    Efficient plasmid DNA transfection of embryonic stem cells, mesenchymal stem cells, neural cell lines and the majority of primary cell lines is a current challenge in gene therapy research. Magnetic nanoparticle-based DNA transfection is a gene vectoring technique that is promising because it is capable of outperforming most other non-viral transfection methods in terms of both transfection efficiency and cell viability. The nature of the DNA vector implemented depends on the target cell phenotype, where the particle surface chemistry and DNA binding/unbinding kinetics of the DNA carrier molecule play a critical role in the many steps required for successful gene transfection. Accordingly, Neuromag, an iron oxide/polymer nanoparticle optimized for transfection of neural phenotypes, outperforms many other nanoparticles and lipidbased DNA carriers. Up to now, improvements to nanomagnetic transfection techniques have focused mostly on particle functionalization and transfection parameter optimization (cell confluence, growth media, serum starvation, magnet oscillation parameters, etc.). None of these parameters are capable of assisting the nuclear translocation of delivered plasmid DNA once the particle-DNA complex is released from the endosome and dissociates in the cell's cytoplasm. In this study, incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid DNA confers improved nuclear translocation, demonstrating significant improvement in nanomagnetic transfection efficiency in differentiated SH-SY5Y neuroblastoma cells. Other parameters, such as days in vitro, are also found to play a role and represent potential targets for further optimization.

  16. Magnetic nanoparticles as gene delivery agents: enhanced transfection in the presence of oscillating magnet arrays

    NASA Astrophysics Data System (ADS)

    McBain, S. C.; Griesenbach, U.; Xenariou, S.; Keramane, A.; Batich, C. D.; Alton, E. W. F. W.; Dobson, J.

    2008-10-01

    Magnetic nanoparticle-based gene transfection has been shown to be effective in combination with both viral vectors and with non-viral agents. In these systems, therapeutic or reporter genes are attached to magnetic nanoparticles which are then focused to the target site/cells via high-field/high-gradient magnets. The technique has been shown to be efficient and rapid for in vitro transfection and compares well with cationic lipid-based reagents, producing good overall transfection levels with lower doses and shorter transfection times. In spite of its potential advantages (particularly for in vivo targeting), the overall transfection levels do not generally exceed those of other non-viral agents. In order to improve the overall transfection levels while maintaining the advantages inherent in this technique, we have developed a novel, oscillating magnet array system which adds lateral motion to the particle/gene complex in order to promote transfection. Experimental results indicate that the system significantly enhances overall in vitro transfection levels in human airway epithelial cells compared to both static field techniques (p<0.005) and the cationic lipids (p<0.001) tested. In addition, it has the previously demonstrated advantages of magnetofection—rapid transfection times and requiring lower levels of DNA than cationic lipid-based transfection agents. This method shows potential for non-viral gene delivery both in vitro and in vivo.

  17. An evaluation and parameterization of stably stratified turbulence: Insights on the atmospheric boundary layer and implications for wind energy

    NASA Astrophysics Data System (ADS)

    Wilson, Jordan M.

    This research focuses on the dynamics of turbulent mixing under stably stratified flow conditions. Velocity fluctuations and instabilities are suppressed by buoyancy forces limiting mixing as stability increases and turbulence decreases until the flow relaminarizes. Theories that ubiquitously assume turbulence collapse above a critical value of the gradient Richardson number (e.g. Ri > Ric) are common in meteorological and oceanographic communities. However, most theories were developed from results of small-scale laboratory and numerical experiments with energetic levels several orders of magnitude less than geophysical flows. Geophysical flows exhibit strong turbulence that enhances the transport of momentum and scalars. The mixing length for the turbulent momentum field, L M, serves as a key parameter in assessing large-scale, energy-containing motions. For a stably stratified turbulent shear flow, the shear production of turbulent kinetic energy, P, is here considered to be of greater relevance than the dissipation rate of turbulent kinetic energy, epsilon. Thus, the turbulent Reynolds number can be recast as Re ≡ k2/(nuP) where k is the turbulent kinetic energy, allowing for a new perspective on flow energetics. Using an ensemble data set of high quality direct numerical simulation (DNS) results, large-eddy simulation (LES) results, laboratory experiments, and observational field data of the stable atmospheric boundary layer (SABL), the dichotomy of data becomes apparent. High mixing rates persist to strong stability (e.g. Ri ≈ 10) in the SABL whereas numerical and laboratory results confirm turbulence collapse for Ri ˜ O(1). While this behavior has been alluded to in literature, this direct comparison of data elucidates the disparity in universal theories of stably stratified turbulence. From this theoretical perspective, a Reynolds-averaged framework is employed to develop and evaluate parameterizations of turbulent mixing based on the competing forces

  18. A portable toxicity biosensor using freeze-dried recombinant bioluminescent bacteria.

    PubMed

    Choi, Sue Hyung; Gu, Man Bock

    2002-05-01

    A portable biosensor has been developed to meet the demands of field toxicity analysis. This biosensor consists of three parts, a freeze-dried biosensing strain within a vial, a small light-proof test chamber, and an optic-fiber connected between the sample chamber and a luminometer. Various genetically engineered bioluminescent bacteria were freeze-dried to measure different types of toxicity based upon their modes of action. GC2 (lac::luxCDABE), a constitutively bioluminescent strain, was used to monitor the general toxicity of samples through a decrease in its bioluminescence, while specific toxicity was detected through the use of strains such as DPD2540 (fabA::luxCDABE), TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2511 (katG::luxCDABE). These inducible strains show an increase in bioluminescence under specific stressful conditions, i.e. membrane-, protein-, DNA-, and oxidative-stress, respectively. The toxicity of a sample could be detected by measuring the bioluminescence 30 min after addition to the freeze-dried strains. In an attempt to enhance the sensitivity of the freeze-dried cells, glucose and Tween 80 were tested as additives. It was found that the addition of glucose had a negative effect on the viability of the freeze-dried cells, while samples having Tween 80 showed an increase in their viability. On the other hand, the addition of either Tween 80 or glucose decreased the final bioluminescent response of DPD2540 in response to 4-chlorophenol. Using these strains, many different chemicals were tested and characterized. This portable biosensor, with a very simple protocol, can be used for field sample analysis and the monitoring of various water systems on-site.

  19. Photon hunting in the twilight zone: visual features of mesopelagic bioluminescent sharks.

    PubMed

    Claes, Julien M; Partridge, Julian C; Hart, Nathan S; Garza-Gisholt, Eduardo; Ho, Hsuan-Ching; Mallefet, Jérôme; Collin, Shaun P

    2014-01-01

    The mesopelagic zone is a visual scene continuum in which organisms have developed various strategies to optimize photon capture. Here, we used light microscopy, stereology-assisted retinal topographic mapping, spectrophotometry and microspectrophotometry to investigate the visual ecology of deep-sea bioluminescent sharks [four etmopterid species (Etmopterus lucifer, E. splendidus, E. spinax and Trigonognathus kabeyai) and one dalatiid species (Squaliolus aliae)]. We highlighted a novel structure, a translucent area present in the upper eye orbit of Etmopteridae, which might be part of a reference system for counterillumination adjustment or acts as a spectral filter for camouflage breaking, as well as several ocular specialisations such as aphakic gaps and semicircular tapeta previously unknown in elasmobranchs. All species showed pure rod hexagonal mosaics with a high topographic diversity. Retinal specialisations, formed by shallow cell density gradients, may aid in prey detection and reflect lifestyle differences; pelagic species display areae centrales while benthopelagic and benthic species display wide and narrow horizontal streaks, respectively. One species (E. lucifer) displays two areae within its horizontal streak that likely allows detection of conspecifics' elongated bioluminescent flank markings. Ganglion cell topography reveals less variation with all species showing a temporal area for acute frontal binocular vision. This area is dorsally extended in T. kabeyai, allowing this species to adjust the strike of its peculiar jaws in the ventro-frontal visual field. Etmopterus lucifer showed an additional nasal area matching a high rod density area. Peak spectral sensitivities of the rod visual pigments (λmax) fall within the range 484-491 nm, allowing these sharks to detect a high proportion of photons present in their habitat. Comparisons with previously published data reveal ocular differences between bioluminescent and non-bioluminescent deep

  20. Numerical modeling of the dynamic response of a bioluminescent bacterial biosensor.

    PubMed

    Affi, Mahmoud; Solliec, Camille; Legentilhomme, Patrick; Comiti, Jacques; Legrand, Jack; Jouanneau, Sulivan; Thouand, Gérald

    2016-12-01

    Water quality and water management are worldwide issues. The analysis of pollutants and in particular, heavy metals, is generally conducted by sensitive but expensive physicochemical methods. Other alternative methods of analysis, such as microbial biosensors, have been developed for their potential simplicity and expected moderate cost. Using a biosensor for a long time generates many changes in the growth of the immobilized bacteria and consequently alters the robustness of the detection. This work simulated the operation of a biosensor for the long-term detection of cadmium and improved our understanding of the bioluminescence reaction dynamics of bioreporter bacteria inside an agarose matrix. The choice of the numerical tools is justified by the difficulty to measure experimentally in every condition the biosensor functioning during a long time (several days). The numerical simulation of a biomass profile is made by coupling the diffusion equation and the consumption/reaction of the nutrients by the bacteria. The numerical results show very good agreement with the experimental profiles. The growth model verified that the bacterial growth is conditioned by both the diffusion and the consumption of the nutrients. Thus, there is a high bacterial density in the first millimeter of the immobilization matrix. The growth model has been very useful for the development of the bioluminescence model inside the gel and shows that a concentration of oxygen greater than or equal to 22 % of saturation is required to maintain a significant level of bioluminescence. A continuous feeding of nutrients during the process of detection of cadmium leads to a biofilm which reduces the diffusion of nutrients and restricts the presence of oxygen from the first layer of the agarose (1 mm) and affects the intensity of the bioluminescent reaction. The main advantage of this work is to link experimental works with numerical models of growth and bioluminescence in order to provide a

  1. Photon Hunting in the Twilight Zone: Visual Features of Mesopelagic Bioluminescent Sharks

    PubMed Central

    Claes, Julien M.; Partridge, Julian C.; Hart, Nathan S.; Garza-Gisholt, Eduardo; Ho, Hsuan-Ching; Mallefet, Jérôme; Collin, Shaun P.

    2014-01-01

    The mesopelagic zone is a visual scene continuum in which organisms have developed various strategies to optimize photon capture. Here, we used light microscopy, stereology-assisted retinal topographic mapping, spectrophotometry and microspectrophotometry to investigate the visual ecology of deep-sea bioluminescent sharks [four etmopterid species (Etmopterus lucifer, E. splendidus, E. spinax and Trigonognathus kabeyai) and one dalatiid species (Squaliolus aliae)]. We highlighted a novel structure, a translucent area present in the upper eye orbit of Etmopteridae, which might be part of a reference system for counterillumination adjustment or acts as a spectral filter for camouflage breaking, as well as several ocular specialisations such as aphakic gaps and semicircular tapeta previously unknown in elasmobranchs. All species showed pure rod hexagonal mosaics with a high topographic diversity. Retinal specialisations, formed by shallow cell density gradients, may aid in prey detection and reflect lifestyle differences; pelagic species display areae centrales while benthopelagic and benthic species display wide and narrow horizontal streaks, respectively. One species (E. lucifer) displays two areae within its horizontal streak that likely allows detection of conspecifics' elongated bioluminescent flank markings. Ganglion cell topography reveals less variation with all species showing a temporal area for acute frontal binocular vision. This area is dorsally extended in T. kabeyai, allowing this species to adjust the strike of its peculiar jaws in the ventro-frontal visual field. Etmopterus lucifer showed an additional nasal area matching a high rod density area. Peak spectral sensitivities of the rod visual pigments (λmax) fall within the range 484–491 nm, allowing these sharks to detect a high proportion of photons present in their habitat. Comparisons with previously published data reveal ocular differences between bioluminescent and non-bioluminescent deep

  2. Three-dimensional multi bioluminescent sources reconstruction based on adaptive finite element method

    NASA Astrophysics Data System (ADS)

    Ma, Xibo; Tian, Jie; Zhang, Bo; Zhang, Xing; Xue, Zhenwen; Dong, Di; Han, Dong

    2011-03-01

    Among many optical molecular imaging modalities, bioluminescence imaging (BLI) has more and more wide application in tumor detection and evaluation of pharmacodynamics, toxicity, pharmacokinetics because of its noninvasive molecular and cellular level detection ability, high sensitivity and low cost in comparison with other imaging technologies. However, BLI can not present the accurate location and intensity of the inner bioluminescence sources such as in the bone, liver or lung etc. Bioluminescent tomography (BLT) shows its advantage in determining the bioluminescence source distribution inside a small animal or phantom. Considering the deficiency of two-dimensional imaging modality, we developed three-dimensional tomography to reconstruct the information of the bioluminescence source distribution in transgenic mOC-Luc mice bone with the boundary measured data. In this paper, to study the osteocalcin (OC) accumulation in transgenic mOC-Luc mice bone, a BLT reconstruction method based on multilevel adaptive finite element (FEM) algorithm was used for localizing and quantifying multi bioluminescence sources. Optical and anatomical information of the tissues are incorporated as a priori knowledge in this method, which can reduce the ill-posedness of BLT. The data was acquired by the dual modality BLT and Micro CT prototype system that was developed by us. Through temperature control and absolute intensity calibration, a relative accurate intensity can be calculated. The location of the OC accumulation was reconstructed, which was coherent with the principle of bone differentiation. This result also was testified by ex vivo experiment in the black 96-plate well using the BLI system and the chemiluminescence apparatus.

  3. Clickable Poly(ionic liquids): A Materials Platform for Transfection.

    PubMed

    Freyer, Jessica L; Brucks, Spencer D; Gobieski, Graham S; Russell, Sebastian T; Yozwiak, Carrie E; Sun, Mengzhen; Chen, Zhixing; Jiang, Yivan; Bandar, Jeffrey S; Stockwell, Brent R; Lambert, Tristan H; Campos, Luis M

    2016-09-26

    The potential applications of cationic poly(ionic liquids) range from medicine to energy storage, and the development of efficient synthetic strategies to target innovative cationic building blocks is an important goal. A post-polymerization click reaction is reported that provides facile access to trisaminocyclopropenium (TAC) ion-functionalized macromolecules of various architectures, which are the first class of polyelectrolytes that bear a formal charge on carbon. Quantitative conversions of polymers comprising pendant or main-chain secondary amines were observed for an array of TAC derivatives in three hours using near equimolar quantities of cyclopropenium chlorides. The resulting TAC polymers are biocompatible and efficient transfection agents. This robust, efficient, and orthogonal click reaction of an ionic liquid, which we term ClickabIL, allows straightforward screening of polymeric TAC derivatives. This platform provides a modular route to synthesize and study various properties of novel TAC-based polymers.

  4. DNA-poly(diallyldimethylammonium chloride) complexation and transfection efficiency.

    PubMed

    Alatorre-Meda, Manuel; Taboada, Pablo; Krajewska, Barbara; Willemeit, Markus; Deml, Alexander; Klösel, Roland; Rodríguez, Julio R

    2010-07-29

    The present work assesses the influence of the cationic charge density (CD) and the cationic valence of poly(diallyldimethylammonium chloride) (pDADMAC) on the DNA compaction and subsequent transfection. Four homopolymers (CD = 1, with different valences) and one copolymer, poly(acrylamide-co-diallyldimethylammonium chloride) (coDADMAC) (CD < 1, equivalent in valence to one of the homopolymers), were studied. The characterization of the DNA-pDADMAC complexes (polyplexes) as a function of the polycation nitrogen to DNA phosphate molar ratios, N/P, was done by means of conductometry, electrophoretic mobility (zeta-potential), dynamic light scattering (DLS), isothermal titration calorimetry (ITC), atomic force microscopy (AFM), and beta-galactosidase (ONPG) and luciferase expression assays at 25 degrees C and physiological pH. In general, all polyplexes rendered compact and stable structures (R(H) approximately 100 nm) with positive surface charges ( approximately 11 mV) but low transfection efficiencies. As revealed by ITC, the DNA-pDADMAC complexation was characterized by a high binding affinity, the process being entropically driven. In particular, two characteristic ratios ((N/P)c and (N/P)*) were detected. Conductometry and ITC data demonstrated that the DNA compaction ratio, (N/P)c, was mainly governed by CD. Meanwhile the ratio from which the polyplex size remained constant, (N/P)*, was found to be valence-dependent as revealed by DLS. On the other hand, the low transfer rate of the polyplexes appeared to be correlated with the high binding affinity observed throughout the complexation process and with a core-shell structure the complexes presumably adopt.

  5. The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines.

    PubMed

    Regla-Nava, Jose A; Jimenez-Guardeño, Jose M; Nieto-Torres, Jose L; Gallagher, Thomas M; Enjuanes, Luis; DeDiego, Marta L

    2013-11-01

    Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis.

  6. Theoretical tuning of the firefly bioluminescence spectra by the modification of oxyluciferin

    NASA Astrophysics Data System (ADS)

    Cheng, Yuan-Yuan; Zhu, Jia; Liu, Ya-Jun

    2014-01-01

    Extending the firefly bioluminescence is of practical significance for the improved visualization of living cells and the development of a multicolor reporter. Tuning the color of bioluminescence in fireflies mainly involves the modification of luciferase and luciferin. In this Letter, we theoretically studied the emission spectra of 9 firefly oxyluciferin analogs in the gas phase and in solutions. Three density functionals, including B3LYP, CAM-B3LYP and M06-2X, were employed to theoretically predict the efficiently luminescent analogs. The reliable functionals for calculating the targeted systems were suggested. The luminescence efficiency, solvent effects, and substituent effects are discussed based on the calculated results.

  7. A bioluminescent arsenite biosensor designed for inline water analyzer.

    PubMed

    Prévéral, Sandra; Brutesco, Catherine; Descamps, Elodie C T; Escoffier, Camille; Pignol, David; Ginet, Nicolas; Garcia, Daniel

    2017-01-01

    Whole-cell biosensors based on the reporter gene system can offer rapid detection of trace levels of organic or metallic compounds in water. They are well characterized in laboratory conditions, but their transfer into technological devices for the surveillance of water networks remains at a conceptual level. The development of a semi-autonomous inline water analyzer stumbles across the conservation of the bacterial biosensors over a period of time compatible with the autonomy requested by the end-user while maintaining a satisfactory sensitivity, specificity, and time response. We focused here on assessing the effect of lyophilization on two biosensors based on the reporter gene system and hosted in Escherichia coli. The reporter gene used here is the entire bacterial luciferase lux operon (luxCDABE) for an autonomous bioluminescence emission without the need to add any substrate. In the cell-survival biosensor that is used to determine the overall fitness of the bacteria when mixed with the water sample, lux expression is driven by a constitutive E. coli promoter PrpoD. In the arsenite biosensor, the arsenite-inducible promoter P ars involved in arsenite resistance in E. coli controls lux expression. Evaluation of the shelf life of these lyophilized biosensors kept at 4 °C over a year evidenced that about 40 % of the lyophilized cells can be revived in such storage conditions. The performances of the lyophilized biosensor after 7 months in storage are maintained, with a detection limit of 0.2 μM arsenite for a response in about an hour with good reproducibility. These results pave the way to the use in tandem of both biosensors (one for general toxicity and one for arsenite contamination) as consumables of an autonomous analyzer in the field.

  8. Application of fluorescence spectroscopy and multispectral imaging for non-invasive estimation of GFP transfection efficiency

    NASA Astrophysics Data System (ADS)

    Tamošiūnas, M.; Jakovels, D.; Lihačovs, A.; Kilikevičius, A.; Baltušnikas, J.; Kadikis, R.; Šatkauskas, S.

    2014-10-01

    Electroporation and ultrasound induced sonoporation has been showed to induce plasmid DNA transfection to the mice tibialis cranialis muscle. It offers new prospects for gene therapy and cancer treatment. However, numerous experimental data are still needed to deliver the plausible explanation of the mechanisms governing DNA electro- or sono-transfection, as well as to provide the updates on transfection protocols for transfection efficiency increase. In this study we aimed to apply non-invasive optical diagnostic methods for the real time evaluation of GFP transfection levels at the reduced costs for experimental apparatus and animal consumption. Our experimental set-up allowed monitoring of GFP levels in live mice tibialis cranialis muscle and provided the parameters for DNA transfection efficiency determination.

  9. Oscillating Magnet Array−Based Nanomagnetic Gene Transfection: A Valuable Tool for Molecular Neurobiology Studies

    PubMed Central

    Subramanian, Mahendran; Tyler, Aimee-Jayne; Luther, Eva Maria; Daniel, Elena Di; Lim, Jenson; Dobson, Jon

    2017-01-01

    To develop treatments for neurodegenerative disorders, it is critical to understand the biology and function of neurons in both normal and diseased states. Molecular studies of neurons involve the delivery of small biomolecules into cultured neurons via transfection to study genetic variants. However, as cultured primary neurons are sensitive to temperature change, stress, and shifts in pH, these factors make biomolecule delivery difficult, particularly non-viral delivery. Herein we used oscillating nanomagnetic gene transfection to successfully transfect SH-SY5Y cells as well as primary hippocampal and cortical neurons on different days in vitro. This novel technique has been used to effectively deliver genetic material into various cell types, resulting in high transfection efficiency and viability. From these observations and other related studies, we suggest that oscillating nanomagnetic gene transfection is an effective method for gene delivery into hard-to-transfect neuronal cell types. PMID:28336862

  10. Metabolic inhibition increases activity of connexin-32 hemichannels permeable to Ca2+ in transfected HeLa cells

    PubMed Central

    Sánchez, Helmuth A.; Orellana, Juan A.; Verselis, Vytas K.

    2009-01-01

    Numerous cell types express functional connexin (Cx) hemichannels (HCs), and membrane depolarization and/or exposure to a divalent cation-free bathing solution (DCFS) have been shown to promote HC opening. However, little is known about conditions that can promote HC opening in the absence of strong depolarization and when extracellular divalent cation concentrations remain at physiological levels. Here the effects of metabolic inhibition (MI), an in vitro model of ischemia, on the activity of mouse Cx32 HCs were examined. In HeLa cells stably transfected with mouse Cx32 (HeLa-Cx32), MI induced an increase in cellular permeability to ethidium (Etd). The increase in Etd uptake was directly related to an increase in levels of Cx32 HCs present at the cell surface. Moreover, MI increased membrane currents in HeLa-Cx32 cells. Underlying these currents were channels exhibiting a unitary conductance of ∼90 pS, consistent with Cx32 HCs. These currents and Etd uptake were blocked by HC inhibitors. The increase in Cx32 HC activity was preceded by a rapid reduction in mitochondrial membrane potential and a rise in free intracellular Ca2+ concentration ([Ca2+]i). The increase in free [Ca2+]i was prevented by HC blockade or exposure to extracellular DCFS and was virtually absent in parental HeLa cells. Moreover, inhibition of Cx32 HCs expressed by HeLa cells in low-confluence cultures drastically reduced cell death induced by oxygen-glucose deprivation, which is a more physiological model of ischemia-reperfusion. Thus HC blockade could reduce the increase in free [Ca2+]i and cell death induced by ischemia-like conditions in cells expressing Cx32 HCs. PMID:19587218

  11. Hypothesis about brilliant lights by bioluminescent photons in near death experiences.

    PubMed

    Bókkon, István; Salari, Vahid

    2012-07-01

    In near death experiences (NDEs), seeing a brilliant light may arise in the recovery period following cardiac arrest, but the subjects can think that these experiences had happened during the actual period itself. Here we hypothesize a biophysical explanation about the encounter with a brilliant light in NDEs. Accordingly, meeting brilliant light in NDEs is due to the reperfusion that induces unregulated overproduction of free radicals and excited biomolecules among them in numerous parts in the visual system. Unregulated free radicals and excited species can produce a transient increase of bioluminescent photons in different areas of the visual system. If this excess of bioluminescent photon emission exceeds a threshold, they can appear as (phosphene) lights in our mind. In other words, seeing a brilliant light in NDEs may due to bioluminescent photons simultaneously generated in the recovery phase of numerous areas of the visual system and the brain interprets these intrinsic bioluminescent photons as if they were originated from the external visual world. Although our biophysical explanation about brilliant light phenomenon in NDEs can be promising, we do not reject further potential notions.

  12. Bioluminescence ATP Monitoring for the Routine Assessment of Food Contact Surface Cleanliness in a University Canteen

    PubMed Central

    Osimani, Andrea; Garofalo, Cristiana; Clementi, Francesca; Tavoletti, Stefano; Aquilanti, Lucia

    2014-01-01

    ATP bioluminescence monitoring and traditional microbiological analyses (viable counting of total mesophilic aerobes, coliforms and Escherichia coli) were used to evaluate the effectiveness of Sanitation Standard Operating Procedures (SSOP) at a university canteen which uses a HACCP-based approach. To that end, 10 cleaning control points (CPs), including food contact surfaces at risk of contamination from product residues or microbial growth, were analysed during an 8-month monitoring period. Arbitrary acceptability limits were set for both microbial loads and ATP bioluminescence readings. A highly significant correlation (r = 0.99) between the means of ATP bioluminescence readings and the viable counts of total mesophilic aerobes was seen, thus revealing a strong association of these parameters with the level of surface contamination. Among CPs, the raw meat and multi-purpose chopping boards showed the highest criticalities. Although ATP bioluminescence technology cannot substitute traditional microbiological analyses for the determination of microbial load on food contact surfaces, it has proved to be a powerful tool for the real time monitoring of surface cleanliness at mass catering plants, for verify the correct application of SSOP, and hence for their implementation/revision in the case of poor hygiene. PMID:25329534

  13. Comparison of the Lumac and Monolight systems for detection of bacteriuria by bioluminescence.

    PubMed Central

    Drow, D L; Baum, C H; Hirschfield, G

    1984-01-01

    The development of practical and rapid methods for detection of infectious-disease-producing agents in clinical specimens is the most important current goal of clinical microbiology. Bioluminescence is a technique which is rapid and potentially sensitive enough to detect significant numbers of bacteria in urine specimens. To determine whether bioluminescence is practical and cost effective for routine use, we compared two commercially available instruments and kits, Lumac and Monolight, to standard bacterial cultures on 986 urine specimens. Lumac had an overall 83.7% agreement with cultures, a sensitivity of 92.4%, and a specificity of 79.4%. Monolight had 83.5% agreement with cultures, a sensitivity of 89.1%, and a specificity of 81.8%. There were 13.8% false-positive results and 2.5% false-negative results with both systems. When only potentially significant organisms were included, the false-negative rate was reduced to ca. 1%. Both systems are sufficiently accurate to be recommended for routine use. The cost of bioluminescence is higher than that of bacterial cultures, and bioluminescence may not be cost effective in some laboratories. PMID:6490862

  14. Enumeration of bacterial cell numbers by amplified firefly bioluminescence without cultivation.

    PubMed

    Sakakibara, Tatsuya; Murakami, Seiji; Imai, Kazuhiro

    2003-01-01

    We recently developed a novel bioluminescent enzymatic cycling assay for ATP and AMP with the concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK), where AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. Background luminescence derived from contaminating ATP and AMP in the reagent was reduced using adenosine phosphate deaminase which degrades ATP, ADP, and AMP, resulting in constant and highly amplified bioluminescence with low background luminescence. To detect bacterial cells without cultivation, we applied the above bioluminescent enzymatic cycling reagent to rapid microbe detection system. ATP spots (0.31-5.0 amol/spot) at the level of a single bacterial cell were detected with 5 min signal integration, signifying that integrated luminescence was amplified 43 times in comparison to traditional ATP bioluminescence. Consequently, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus brevis in beer were detected without cultivation. Significant correlation was observed between the number of signal spots obtained using this novel system and the colony-forming units observed with the conventional colony-counting method (R(2)=0.973).

  15. Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil.

    PubMed

    Amaral, D T; Arnoldi, F G C; Rosa, S P; Viviani, V R

    2014-08-01

    Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae.

  16. Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis.

    PubMed

    Kita-Tsukamoto, Kumiko; Wada, Minoru; Yao, Katomi; Kamiya, Akiko; Yoshizawa, Susumu; Uchiyama, Nami; Kogure, Kazuhiro

    2006-03-01

    To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.

  17. Characteristics analysis of the luzA gene encoding chaperone from Photobacterium leiognathi related to bioluminescence.

    PubMed

    Lin, J W; Lin, B J; Chen, H Y; Weng, S F

    1998-03-27

    Nucleotide sequence of the luzA gene (GenBank accession No. AF039303) from Photobacterium leiognathi ATCC 25521 (NCIMB 2193) has been determined, and the chaperone encoded by the luzA gene was deduced. The LuzA chaperone has a calculated M(r) 26,295 and comprises 230 amino acid residues; the hydrophobic alpha-helix N-terminal 21 amino acid residues MKKTIFALLFMSVFI SYPSFA is the leader peptide, therefore the matured LuzA chaperone has a calculated M(r) 23,871 and comprises 209 amino acid residues only. The periplasmic LuzA chaperone is the protein concerned with the protein folding, assembly and stability. The luzA gene and the related genes are closely linked to the sod gene, that encoding Cu/Zn superoxide dismutase enables to enhance bioluminescence of the lux operon; the gene order of the luzA gene and related genes is -ufo'-luzA-ufoI-ufoII-ter->-R&R'-sod-ufo-- >. In trans complementation bioluminoassays in vivo elicit that the LuzA chaperone might be not directly concerned with bioluminescence of the lux operon from P. leiognathi in E. coli, but might enable to stabilize the proteins related to bioluminescence. The unidentified ufoII gene closely linked to the luzA gene is able to enhance bioluminescence.

  18. Purification of lumazine proteins from Photobacterium leiognathi and Photobacterium phosphoreum: bioluminescence properties.

    PubMed

    O'Kane, D J; Karle, V A; Lee, J

    1985-03-12

    Bright strains of the marine bioluminescent bacterium Photobacterium leiognathi produce a "lumazine protein" in amounts comparable to that previously found in Photobacterium phosphoreum. New protocols are developed for the purification to homogeneity of the proteins from both species in yields up to 60%. In dimmer strains the amounts of lumazine protein in extracts are less, and also there is an accompanying shift of the bioluminescence spectral maximum to longer wavelength, 492 nm. Both types of lumazine proteins have identical fluorescence spectra, with maxima at 475 nm, so it is suggested that, whereas lumazine protein is the major emitter in bright strains, there is a second emitter also present with a fluorescence maximum at longer wavelength. The two species of lumazine protein have the same 276 nm/visible absorbance ratio, 2.2, but differ in visible maxima: P. phosphoreum, 417 nm; P. leiognathi, 420 nm. For the latter the bound lumazine has epsilon 420 = 10 100 M-1 cm-1, practically the same as in free solution. The two lumazine proteins also differ quantitatively in their effect on the in vitro bioluminescence reaction, i.e., at blue shifting the bioluminescence spectrum or altering the kinetics. The P. phosphoreum lumazine protein is more effective with its homologous luciferase or with P. leiognathi luciferase than is the lumazine protein from P. leiognathi. These differences may have an electrostatic origin.

  19. Monitoring the Response of Hyperbilirubinemia in the Mouse Brain by In Vivo Bioluminescence Imaging.

    PubMed

    Manni, Isabella; Di Rocco, Giuliana; Fusco, Salvatore; Leone, Lucia; Barbati, Saviana Antonella; Carapella, Carmine Maria; Grassi, Claudio; Piaggio, Giulia; Toietta, Gabriele

    2016-12-28

    Increased levels of unconjugated bilirubin are neurotoxic, but the mechanism leading to neurological damage has not been completely elucidated. Innovative strategies of investigation are needed to more precisely define this pathological process. By longitudinal in vivo bioluminescence imaging, we noninvasively visualized the brain response to hyperbilirubinemia in the MITO-Luc mouse, in which light emission is restricted to the regions of active cell proliferation. We assessed that acute hyperbilirubinemia promotes bioluminescence in the brain region, indicating an increment in the cell proliferation rate. Immunohistochemical detection in brain sections of cells positive for both luciferase and the microglial marker allograft inflammatory factor 1 suggests proliferation of microglial cells. In addition, we demonstrated that brain induction of bioluminescence was altered by pharmacological displacement of bilirubin from its albumin binding sites and by modulation of the blood-brain barrier permeability, all pivotal factors in the development of bilirubin-induced neurologic dysfunction. We also determined that treatment with minocycline, an antibiotic with anti-inflammatory and neuroprotective properties, or administration of bevacizumab, an anti-vascular endothelial growth factor antibody, blunts bilirubin-induced bioluminescence. Overall the study supports the use of the MITO-Luc mouse as a valuable tool for the rapid response monitoring of drugs aiming at preventing acute bilirubin-induced neurological dysfunction.

  20. Detection of light and vibration modulates bioluminescence intensity in the glowworm, Arachnocampa flava.

    PubMed

    Mills, Rebecca; Popple, Julie-Anne; Veidt, Martin; Merritt, David John

    2016-04-01

    Glowworms are larval fungus gnats that emit light from a specialised abdominal light organ. The light attracts small arthropod prey to their web-like silk snares. Larvae glow throughout the night and can modulate their bioluminescence in response to sensory input. To better understand light output regulation and its ecological significance, we examined the larvae's reaction to light exposure, vibration and sound. Exposure to a 5-min light pulse in the laboratory causes larvae to exponentially decrease their light output over 5-10 min until they completely switch off. They gradually return to pre-exposure levels but do not show a rebound. Larvae are most sensitive to ultraviolet light, then blue, green and red. Vibration of the larval snares results in a several-fold increase in bioluminescence over 20-30 s, followed by an exponential return to pre-exposure levels over 15-30 min. Under some conditions, larvae can respond to vibration by initiating bioluminescence when they are not glowing; however, the response is reduced compared to when they are glowing. We propose that inhibitory and excitatory mechanisms combine to modulate bioluminescence intensity by regulating biochemical reactions or gating the access of air to the light organ.

  1. Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift.

    PubMed

    Kheirabadi, Mitra; Sharafian, Zohreh; Naderi-Manesh, Hossein; Heineman, Udo; Gohlke, Ulrich; Hosseinkhani, Saman

    2013-12-01

    Firefly bioluminescence reaction in the presence of Mg(2+), ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350-359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7Å and 2.2Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351-359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission.

  2. Bioluminophore and Flavin Mononucleotide Fluorescence Quenching of Bacterial Bioluminescence-A Theoretical Study.

    PubMed

    Luo, Yanling; Liu, Ya-Jun

    2016-11-02

    Bacterial bioluminescence with continuous glow has been applied to the fields of environmental toxin monitoring, drug screening, and in vivo imaging. Nonetheless, the chemical form of the bacterial bioluminophore is still a bone of contention. Flavin mononucleotide (FMN), one of the light-emitting products, and 4a-hydroxy-5-hydro flavin mononucleotide (HFOH), an intermediate of the chemical reactions, have both been assumed candidates for the light emitter because they have similar molecular structures and fluorescence wavelengths. The latter is preferred in experiments and was assigned in our previous density functional study. HFOH displays weak fluorescence in solutions, but exhibits strong bioluminescence in the bacterial luciferase. FMN shows the opposite behavior; its fluorescence is quenched when it is bound to the luciferase. This is the first example of flavin fluorescence quenching observed in bioluminescent systems and is merely an observation, both the quenching mechanism and quencher are still unclear. Based on theoretical analysis of high-level quantum mechanics (QM), combined QM and molecular mechanics (QM/MM), and molecular dynamics (MD), this paper confirms that HFOH in its first singlet excited state is the bioluminophore of bacterial bioluminescence. More importantly, the computational results indicate that Tyr110 in the luciferase quenches the FMN fluorescence via an electron-transfer mechanism.

  3. Total evidence phylogeny and the evolution of adult bioluminescence in fireflies (Coleoptera: Lampyridae).

    PubMed

    Martin, Gavin J; Branham, Marc A; Whiting, Michael F; Bybee, Seth M

    2017-02-01

    Fireflies are some of the most captivating organisms on the planet. They have a rich history as subjects of scientific study, especially in relation to their bioluminescent behavior. Yet, the phylogenetic relationships of fireflies are still poorly understood. Here, we present the first total evidence approach to reconstruct lampyrid phylogeny using both a molecular matrix from six loci and an extensive morphological matrix. Using this phylogeny we test the hypothesis that adult bioluminescence evolved after the origin of the firefly clade. The ancestral state of adult bioluminescence is recovered as non-bioluminescent with one to six gains and five to ten subsequent losses. The monophyly of the family, as well as the subfamilies is also tested. Ototretinae, Cyphonocerinae, Luciolinae (incl. Pristolycus), Amydetinae, "cheguevarinae" sensu Jeng 2008, and Photurinae are highly supported as monophyletic. With the exception of four taxa, Lampyrinae is also recovered as monophyletic with high support. Based on phylogenetic and morphological data Lamprohiza, Phausis, and Lamprigera are transferred to Lampyridae incertae sedis.

  4. A Bioluminescence Assay Using Nitrosomonas europaea for Rapid and Sensitive Detection of Nitrification Inhibitors

    PubMed Central

    Iizumi, Taro; Mizumoto, Masahiro; Nakamura, Kanji

    1998-01-01

    An expression vector for the luxAB genes, derived from Vibrio harveyi, was introduced into Nitrosomonas europaea. Although the recombinant strain produced bioluminescence due to the expression of the luxAB genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin. When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity. The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O2 uptake and NO2− production rates. The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase. These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways. We conclude that the assay system using luminous Nitrosomonas can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants. PMID:9758781

  5. Polyphyly of non-bioluminescent Vibrio fischeri sharing a lux-locus deletion.

    PubMed

    Wollenberg, M S; Preheim, S P; Polz, M F; Ruby, E G

    2012-03-01

    This study reports the first description and molecular characterization of naturally occurring, non-bioluminescent strains of Vibrio fischeri. These 'dark' V. fischeri strains remained non-bioluminescent even after treatment with both autoinducer and aldehyde, substrate additions that typically maximize light production in dim strains of luminous bacteria. Surprisingly, the entire lux locus (eight genes) was absent in over 97% of these dark V. fischeri strains. Although these strains were all collected from a Massachusetts (USA) estuary in 2007, phylogenetic reconstructions allowed us to reject the hypothesis that these newly described non-bioluminescent strains exhibit monophyly within the V. fischeri clade. These dark strains exhibited a competitive disadvantage against native bioluminescent strains when colonizing the light organ of the model V. fischeri host, the Hawaiian bobtail squid Euprymna scolopes. Significantly, we believe that the data collected in this study may suggest the first observation of a functional, parallel locus-deletion event among independent lineages of a non-pathogenic bacterial species.

  6. Real time detection of live microbes using a highly sensitive bioluminescent nitroreductase probe.

    PubMed

    Wong, Roger H F; Kwong, Thomas; Yau, Kwok-Hei; Au-Yeung, Ho Yu

    2015-03-14

    A highly sensitive and selective nitroreductase probe, showing a rapid and strong bioluminescence enhancement (>100-fold in 5 minutes), and its initial application in the real time detection of both Gram positive and Gram negative live bacteria and monitoring of their growth has been reported.

  7. Bioluminescence ATP monitoring for the routine assessment of food contact surface cleanliness in a university canteen.

    PubMed

    Osimani, Andrea; Garofalo, Cristiana; Clementi, Francesca; Tavoletti, Stefano; Aquilanti, Lucia

    2014-10-17

    ATP bioluminescence monitoring and traditional microbiological analyses (viable counting of total mesophilic aerobes, coliforms and Escherichia coli) were used to evaluate the effectiveness of Sanitation Standard Operating Procedures (SSOP) at a university canteen which uses a HACCP-based approach. To that end, 10 cleaning control points (CPs), including food contact surfaces at risk of contamination from product residues or microbial growth, were analysed during an 8-month monitoring period. Arbitrary acceptability limits were set for both microbial loads and ATP bioluminescence readings. A highly significant correlation (r = 0.99) between the means of ATP bioluminescence readings and the viable counts of total mesophilic aerobes was seen, thus revealing a strong association of these parameters with the level of surface contamination. Among CPs, the raw meat and multi-purpose chopping boards showed the highest criticalities. Although ATP bioluminescence technology cannot substitute traditional microbiological analyses for the determination of microbial load on food contact surfaces, it has proved to be a powerful tool for the real time monitoring of surface cleanliness at mass catering plants, for verify the correct application of SSOP, and hence for their implementation/revision in the case of poor hygiene.

  8. Monitoring the Response of Hyperbilirubinemia in the Mouse Brain by In Vivo Bioluminescence Imaging

    PubMed Central

    Manni, Isabella; Di Rocco, Giuliana; Fusco, Salvatore; Leone, Lucia; Barbati, Saviana Antonella; Carapella, Carmine Maria; Grassi, Claudio; Piaggio, Giulia; Toietta, Gabriele

    2016-01-01

    Increased levels of unconjugated bilirubin are neurotoxic, but the mechanism leading to neurological damage has not been completely elucidated. Innovative strategies of investigation are needed to more precisely define this pathological process. By longitudinal in vivo bioluminescence imaging, we noninvasively visualized the brain response to hyperbilirubinemia in the MITO-Luc mouse, in which light emission is restricted to the regions of active cell proliferation. We assessed that acute hyperbilirubinemia promotes bioluminescence in the brain region, indicating an increment in the cell proliferation rate. Immunohistochemical detection in brain sections of cells positive for both luciferase and the microglial marker allograft inflammatory factor 1 suggests proliferation of microglial cells. In addition, we demonstrated that brain induction of bioluminescence was altered by pharmacological displacement of bilirubin from its albumin binding sites and by modulation of the blood–brain barrier permeability, all pivotal factors in the development of bilirubin-induced neurologic dysfunction. We also determined that treatment with minocycline, an antibiotic with anti-inflammatory and neuroprotective properties, or administration of bevacizumab, an anti-vascular endothelial growth factor antibody, blunts bilirubin-induced bioluminescence. Overall the study supports the use of the MITO-Luc mouse as a valuable tool for the rapid response monitoring of drugs aiming at preventing acute bilirubin-induced neurological dysfunction. PMID:28036021

  9. Controlled field release of a bioluminescent genetically engineered microorganism for bioremediation process monitoring and control

    SciTech Connect

    Ripp, S.; Nivens, D.E.; Ahn, Y.; Werner, C.; Jarrell, J. IV; Easter, J.P.; Cox, C.D.; Burlage, R.S.; Sayler, G.S.

    2000-03-01

    Pseudomonas fluorescens HK44 represents the first genetically engineered microorganism approved for field testing in the United States for bioremediation purposes. Strain HK44 harbors an introduced lux gene fused within a naphthalene degradative pathway, thereby allowing this recombinant microbe to bioluminescent as it degrades specific polyaromatic hydrocarbons such as naphthalene. The bioremediation process can therefore be monitored by the detection of light. P. fluorescens HK44 was inoculated into the vadose zone of intermediate-scale, semicontained soil lysimeters contaminated with naphthalene, anthracene, and phenanthrene, and the population dynamics were followed over an approximate 2-year period in order to assess the long-term efficacy of using strain HK44 for monitoring and controlling bioremediation processes. Results showed that P. fluorescens HK44 was capable of surviving initial inoculation into both hydrocarbon contaminated and uncontaminated soils and was recoverable from these soils 660 days post inoculation. It was also demonstrated that strain HK44 was capable of generating bioluminescence in response to soil hydrocarbon bioavailability. Bioluminescence approaching 166,000 counts/s was detected in fiber optic-based biosensor devices responding to volatile polyaromatic hydrocarbons, while a portable photomultiplier module detected bioluminescence at an average of 4300 counts/s directly from soil-borne HK44 cells within localized treatment areas. The utilization of lux-based bioreporter microorganisms therefore promises to be a viable option for in situ determination of environmental contaminant bioavailability and biodegradation process monitoring and control.

  10. Kinetic study of trichloroethylene and toluene degradation by a bioluminescent reporter bacterium

    SciTech Connect

    Kelly, C.J.; Sanseverino, J.; Bienkowski, P.R.; Sayler, G.S.

    1995-12-31

    A constructed bioluminescent reporter bacterium, Pseudomonas putida B2, is very briefly described in this paper. The bacterium degrades toluene and trichloroethylene (TCE), and produces light in the presence of toluene. The light response is an indication of cellular viability and expression of the genes encoding toluene and TCE degrading enzymes.

  11. Use of bioluminescence for detection of genetically engineered microorganisms released into the environment. [Xanthomonas campestris

    SciTech Connect

    Shaw, J.J.; Dane, F.; Geiger, D.; Kloepper, J.W. )

    1992-01-01

    The persistence and movement of strain JS414 of Xanthomonas campestris pv. campestris, which was genetically engineered to bioluminesce, were monitored during a limited field introduction. Bioluminescence and traditional dilution plate counts were determined. Strain JS414 was applied to cabbage plants and surrounding soil by mist inoculation, by wound inoculation, by scattering infested debris among plants, and by incorporating bacteria into the soil. Bioluminescent X. campestris pv. campestris was detected in plant samples and in the rhizosphere up to 6 weeks after inoculation. Movement to uninoculated plants was detected on one occasion, but movement from the immediate release area was not detected. Strain JS414 was detected in soil samples beneath mist- and wound-inoculated plants only at intentionally infested locations and in aerial samples only on the day of inoculation. The authors bioluminescence methods proved to be as sensitive as plating methods for detecting the genetically engineered microorganisms in environmental samples. Their results demonstrate that transgenic incorporation of the luxCDABE operon provides a non-labor-intensive, sensitive detection method for monitoring genetically engineered microorganisms in nature.

  12. Adenosine triphosphate bioluminescence analysis for rapid screening of microbial contamination in non-sterile pharmaceutical samples.

    PubMed

    Jimenez, Luis

    2004-01-01

    An Adenosine Triphosphate (ATP) bioluminescence system was compared and validated against standard methods for rapid microbiological monitoring of several non-sterile pharmaceutical formulations such as creams, tablets, and capsules. Results obtained using 1%, 2.5%, and 10% of product suspensions indicated that most samples that did not contain non-microbial ATP neither inhibited the bioluminescence reaction nor did something else. Ten percent product suspensions were inoculated with different concentrations of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Candida albicans, and Aspergillus niger. Samples were incubated for 24-120 h at 35 degrees C with shaking. Results indicated a strong inhibitory effect of microbial growth, as no microorganisms were detected by using the ATP bioluminescence assay. However, when 1% and 2.5% product suspensions were spiked with the same microorganisms, positive detection was confirmed. After incubation, all microorganisms were detected by the bioluminescence system within 24-72 h. All positive samples were confirmed by using standard plating media. However, to optimize detection of all microorganisms, different enrichment media were developed.

  13. Design, synthesis, and transfection biology of novel cationic glycolipids for use in liposomal gene delivery.

    PubMed

    Banerjee, R; Mahidhar, Y V; Chaudhuri, A; Gopal, V; Rao, N M

    2001-11-22

    The molecular structure of the cationic lipids used in gene transfection strongly influences their transfection efficiency. High transfection efficiencies of non-glycerol-based simple monocationic transfection lipids with hydroxyethyl headgroups recently reported by us (Banerjee et al. J. Med. Chem. 1999, 42, 4292-4299) are consistent with the earlier observations that the presence of hydroxyl functionalities in the headgroup region of a cationic lipid contributes favorably in liposomal gene delivery. Using simple sugar molecules as the source of multiple hydroxyl functionalities in the headgroup region of the transfection lipids, we have synthesized four novel simple monocationic transfection lipids, namely, 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol (1), 1-deoxy-1-[methyl(ditetradecyl)ammonio]-D-arabinitol (2), 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol (3) and 1-deoxy-1-[methyl(dioctadecyl)ammonio]-D-arabinitol (4), containing hydrophobic aliphatic tails and the hydrophilic arabinosyl or xylose sugar groups linked directly to the positively charged nitrogen atom. Syntheses, chemical characterizations, and the transfection biology of these novel transfection lipids 1-4 are described in this paper. Lipid 1, the xylosyl derivative, showed maximum transfection on COS-1 cells. All the lipids showed transfection with cholesterol as colipid and not with dioleoylphosphatidylethanolamine (DOPE). Radioactive quantitation of free and complexed DNA combined with ethidium bromide exclusion measurements suggest that though nearly 70% of the DNA exists as complexed DNA, the DNA may not have condensed as was observed with other cationic lipids. Presence of additional (more than two) hydroxyl functionalities in the headgroup of the cationic lipids appears to have improved the transfection efficiency and made these lipids less cytotoxic compared to two-hydroxyl derivatives.

  14. Bioluminescence imaging of fungal biofilm development in live animals.

    PubMed

    Vande Velde, Greetje; Kucharíková, Soňa; Van Dijck, Patrick; Himmelreich, Uwe

    2014-01-01

    Fungal biofilms formed on various types of medical implants represent a major problem for hospitalized patients. These biofilms and related infections are usually difficult to treat because of their resistance to the classical antifungal drugs. Animal models are indispensable for investigating host-pathogen interactions and for identifying new antifungal targets related to biofilm development. A limited number of animal models is available that can be used for testing novel antifungal drugs in vivo against C. albicans, one of the most common pathogens causing fungal biofilms. Fungal load in biofilms in these models is traditionally analyzed postmortem, requiring host sacrifice and enumeration of microorganisms from individual biofilms in order to evaluate the amount of colony forming units and the efficacy of antifungal treatment. Bioluminescence imaging (BLI) made compatible with small animal models for in vivo biofilm formation is a valuable noninvasive tool to follow-up biofilm development and its treatment longitudinally, reducing the number of animals needed for such studies. Due to the nondestructive and noninvasive nature of BLI, the imaging procedure can be repeated in the same animal, allowing follow-up of the biofilm growth in vivo without removing the implanted device or detaching the biofilm from its substrate. The method descri