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Bioluminescence imaging in mouse models quantifies beta cell mass in the pancreas and after islet transplantation.
Virostko J, Radhika A, Poffenberger G, Chen Z, Brissova M, Gilchrist J, Coleman B, Gannon M, Jansen ED, Powers AC
(2010) Mol Imaging Biol 12: 42-53
MeSH Terms: Animals, Cell Size, Glucose Tolerance Test, Insulin, Insulin-Secreting Cells, Islets of Langerhans Transplantation, Luciferases, Luminescent Measurements, Mice, Models, Animal, Molecular Imaging, Promoter Regions, Genetic, Whole Body Imaging
Show Abstract · Added January 6, 2014
PURPOSE - We developed a mouse model that enables non-invasive assessment of changes in beta cell mass.
PROCEDURES - We generated a transgenic mouse expressing luciferase under control of the mouse insulin I promoter [mouse insulin promoter-luciferase-Vanderbilt University (MIP-Luc-VU)] and characterized this model in mice with increased or decreased beta cell mass and after islet transplantation.
RESULTS - Streptozotocin-induced, diabetic MIP-Luc-VU mice had a progressive decline in bioluminescence that correlated with a decrease in beta cell mass. MIP-Luc-VU animals fed a high-fat diet displayed a progressive increase in bioluminescence that reflected an increase in beta cell mass. MIP-Luc-VU islets transplanted beneath the renal capsule or into the liver emitted bioluminescence proportional to the number of islets transplanted and could be imaged for more than a year.
CONCLUSIONS - Bioluminescence in the MIP-Luc-VU mouse model is proportional to beta cell mass in the setting of increased and decreased beta cell mass and after transplantation.
2 Communities
4 Members
0 Resources
13 MeSH Terms
Role of HSP70 in cellular thermotolerance.
Beckham JT, Wilmink GJ, Mackanos MA, Takahashi K, Contag CH, Takahashi T, Jansen ED
(2008) Lasers Surg Med 40: 704-15
MeSH Terms: Adaptation, Physiological, Animals, Cell Survival, Cells, Cultured, Fibroblasts, HSP70 Heat-Shock Proteins, Hot Temperature, Lasers, Solid-State, Luminescent Measurements, Mice, Models, Biological
Show Abstract · Added August 27, 2013
BACKGROUND AND OBJECTIVE - Thermal pretreatment has been shown to condition tissue to a more severe secondary heat stress. In this research we examined the particular contribution of heat shock protein 70 (HSP70) in thermal preconditioning.
STUDY DESIGN/MATERIALS AND METHODS - For optimization of preshock exposures, a bioluminescent Hsp70-luciferase reporter system in NIH3T3 cells tracked the activation of the Hsp70 gene. Cells in 96-well plates were pretreated in a 43 degrees C water bath for 30 minutes, followed 4 hours later with a severe heat shock at 45 degrees C for 50 minutes. Bioluminescence was measured at 2, 4, 6, 8, and 10 hours after preshock only (PS) and at 4 hours after preshock with heatshock (PS+HS). Viability was assessed 48 hours later with a fluorescent viability dye. Preshock induced thermotolerance was then evaluated in hsp70-containing Murine Embryo Fibroblast (+/+) cells and Hsp70-deficient MEF cells (-/-) through an Arrhenius damage model across varying temperatures (44.5-46 degrees C).
RESULTS - A time gap of 4 hours between preconditioning and the thermal insult was shown to be the most effective for thermotolerance with statistical confidence of P<0.05. The benefit of preshocking was largely abrogated in Hsp70-deficient cells. The Arrhenius data showed that preshocking leads to increases in the activation energies, E(a), and increases in frequency factors, A. The frequency factor increase was significantly greater in Hsp70-deficient cells.
CONCLUSION - The data shows that HSP70 contributes significantly to cellular thermotolerance but there are other pathways that provide residual thermotolerance in cells deficient in Hsp70.
(c) 2008 Wiley-Liss, Inc.
0 Communities
1 Members
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11 MeSH Terms
Comparison of autofluorescence, diffuse reflectance, and Raman spectroscopy for breast tissue discrimination.
Majumder SK, Keller MD, Boulos FI, Kelley MC, Mahadevan-Jansen A
(2008) J Biomed Opt 13: 054009
MeSH Terms: Biomarkers, Tumor, Breast Neoplasms, Female, Humans, Luminescent Measurements, Refractometry, Reproducibility of Results, Sensitivity and Specificity, Spectrometry, Fluorescence, Spectrum Analysis, Raman
Show Abstract · Added February 16, 2016
For a given diagnostic problem, important considerations are the relative performances of the various optical biopsy techniques. A comparative evaluation of fluorescence, diffuse reflectance, combined fluorescence and diffuse reflectance, and Raman spectroscopy in discriminating different histopathologic categories of human breast tissues is reported. Optical spectra were acquired ex vivo from a total of 74 breast tissue samples belonging to 4 distinct histopathologic categories: invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), fibroadenoma (FA), and normal breast tissue. A probability-based multivariate statistical algorithm capable of direct multiclass classification was developed to analyze the diagnostic content of the spectra measured from the same set of breast tissue sites with these different techniques. The algorithm uses the theory of nonlinear maximum representation and discrimination feature for feature extraction, and the theory of sparse multinomial logistic regression for classification. The results reveal that the performance of Raman spectroscopy is superior to that of all others in classifying the breast tissues into respective histopathologic categories. The best classification accuracy was observed to be approximately 99%, 94%, 98%, and 100% for IDC, DCIS, FA, and normal breast tissues, respectively, on the basis of leave-one-sample-out cross-validation, with an overall accuracy of approximately 99%.
0 Communities
1 Members
0 Resources
10 MeSH Terms
Real-time luminescence monitoring of cell-cycle and respiratory oscillations in yeast.
Robertson JB, Stowers CC, Boczko E, Johnson CH
(2008) Proc Natl Acad Sci U S A 105: 17988-93
MeSH Terms: Acetaldehyde, Aerobiosis, Cell Cycle, Ethanol, Genes, Reporter, Luciferases, Luminescent Measurements, Oxygen, Promoter Regions, Genetic, Saccharomyces cerevisiae, Time Factors
Show Abstract · Added February 12, 2015
The use of luciferase reporters has become a precise, noninvasive, high-throughput method for real-time monitoring of promoter activity in living cells, especially for rhythmic biological processes such as circadian rhythms. We developed a destabilized firefly luciferase as a reporter for rhythmic promoter activity in both the cell division and respiratory cycles of the budding yeast Saccharomyces cerevisiae in which real-time luminescence reporters have not been previously applied. The continuous output of light from luciferase reporters allowed us to explore the relationship between the cell division cycle and the yeast respiratory oscillation, including the observation of responses to chemicals that cause phase shifting of the respiratory oscillations. Destabilized firefly luciferase is a good reporter of cell cycle position in synchronized or partially synchronized yeast cultures, in both batch and continuous cultures. In addition, the oxygen dependence of luciferase can be used under certain conditions as a genetically encodable oxygen monitor. Finally, we use this reporter to show that there is a direct correlation between premature induction of cell division and phase resetting of the respiratory oscillation under the continuous culture conditions tested.
0 Communities
1 Members
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11 MeSH Terms
Bioluminescence imaging of vascular endothelial growth factor promoter activity in murine mammary tumorigenesis.
Faley SL, Takahashi K, Crooke CE, Beckham JT, Tomemori T, Shappell SB, Jansen ED, Takahashi T
(2007) Mol Imaging 6: 331-9
MeSH Terms: Animals, Blotting, Western, Female, Green Fluorescent Proteins, Humans, Immunoprecipitation, In Situ Hybridization, Luciferases, Luminescent Measurements, Male, Mammary Neoplasms, Animal, Mice, Mice, Transgenic, Promoter Regions, Genetic, Recombinant Fusion Proteins, Reverse Transcriptase Polymerase Chain Reaction, Vascular Endothelial Growth Factor A
Show Abstract · Added August 27, 2013
Vascular endothelial growth factor (VEGF) is a major inducer of angiogenesis. We generated a transgenic reporter mouse, VEGF-GL, in which an enhanced green fluorescent protein-luciferase fusion protein is expressed under the control of a human VEGF-A promoter. The VEGF-GL mouse exhibited intense bioluminescence throughout the body at 1 week of age. The signals rapidly declined to a relatively low level as the mice grew. The adult VEGF-GL mouse showed restricted bioluminescence to the areas undergoing wound healing. In contrast, the VEGF-GL mice, which were crossed with mouse mammary tumor virus-polyoma virus middle T antigen transgenic mammary tumor mice, exhibited prominent bioluminescence in the tumors, correlating with VEGF transcription. Tumor bioluminescence was observed in the bigenic mice as early as 8 weeks, before tumors were palpable, and the signals increased with tumor growth. In conclusion, the VEGF-GL mouse permits longitudinal and quantitative assessment of VEGF promoter activity in vivo. The model should facilitate understanding of the molecular controls and pathways that regulate VEGF transcription in vivo.
1 Communities
1 Members
0 Resources
17 MeSH Terms
Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues.
Xu X, Soutto M, Xie Q, Servick S, Subramanian C, von Arnim AG, Johnson CH
(2007) Proc Natl Acad Sci U S A 104: 10264-9
MeSH Terms: Animals, Arabidopsis, Arabidopsis Proteins, CCAAT-Enhancer-Binding Proteins, Cell Culture Techniques, Cell Line, Cell Nucleus, Cells, Cultured, Dimerization, Fluorescence Resonance Energy Transfer, Humans, Luminescent Measurements, Luminescent Proteins, Mice, Microscopy, Fluorescence, Pituitary Gland, Plant Proteins, Protein Binding, Proteins, Recombinant Fusion Proteins, Seedlings, Spectrometry, Fluorescence, Tobacco
Show Abstract · Added February 12, 2015
FRET is a well established method for cellular and subcellular imaging of protein interactions. However, FRET obligatorily necessitates fluorescence excitation with its concomitant problems of photobleaching, autofluorescence, phototoxicity, and undesirable stimulation of photobiological processes. A sister technique, bioluminescence resonance energy transfer (BRET), avoids these problems because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effectively in the past. Using a new generation electron bombardment-charge-coupled device camera coupled to an image splitter, we demonstrate that BRET can be used to image protein interactions in plant and animal cells and in tissues; even subcellular imaging is possible. We have applied this technology to image two different protein interactions: (i) dimerization of the developmental regulator, COP1, in plant seedlings; and (ii) CCAAT/enhancer binding protein alpha (C/EBPalpha) in the mammalian nucleus. This advance heralds a host of applications for imaging without fluorescent excitation and its consequent limitations.
0 Communities
1 Members
0 Resources
23 MeSH Terms
Validation of luminescent source reconstruction using single-view spectrally resolved bioluminescence images.
Virostko J, Powers AC, Jansen ED
(2007) Appl Opt 46: 2540-7
MeSH Terms: Algorithms, Animals, Breast, Chickens, Image Enhancement, Image Interpretation, Computer-Assisted, In Vitro Techniques, Luminescent Measurements, Luminescent Proteins, Reproducibility of Results, Sensitivity and Specificity, Spectrometry, Fluorescence
Show Abstract · Added January 20, 2015
We characterize the capabilities and limitations of the Living Image Software 3D Analysis package (Xenogen, Alameda, California) in the reconstruction of calibrated light sources. Sources shallower than the mean free path of light propagation suffered reconstruction inaccuracy. For sources deeper than the mean free path, the average error in depth and intensity reconstruction was less than 4% and 12%, respectively, for homogeneous tissue. The reconstruction of luminescent beads implanted within an optically heterogeneous mouse abdomen proved less accurate. The ability to distinguish multiple sources decreased with increasing source depth. A number of factors influence the accuracy of light source reconstruction.
0 Communities
2 Members
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12 MeSH Terms
Effect of optical tissue clearing on spatial resolution and sensitivity of bioluminescence imaging.
Jansen ED, Pickett PM, Mackanos MA, Virostko J
(2006) J Biomed Opt 11: 041119
MeSH Terms: Administration, Topical, Animals, Dermatologic Agents, Female, Glycerol, Image Enhancement, Luminescent Measurements, Mice, Microscopy, Fluorescence, Reproducibility of Results, Sensitivity and Specificity, Skin
Show Abstract · Added January 20, 2015
In vivo bioluminescence imaging (BLI) is a powerful method of in vivo molecular imaging based on the use of optically active luciferase reporter genes. Although this method provides superior sensitivity relative to other in vivo imaging methods, spatial resolution is poor due to light scattering. The objective of this study was to use hyperosmotic agents to reduce the scattering coefficient and hence improve spatial resolution of the BLI method. A diffusing fiber tip was used to simulate an isotropic point source of bioluminescence emission (550 to 650 nm). Mouse skin was treated in vitro and in vivo with glycerol (50%, 30 min) and measurements of optical properties, and imaging photon counts were made before, during, and after application of glycerol to the skin sample. Glycerol application to mouse skin had little effect on the absorption coefficient but reduced the reduced scattering coefficient by more than one order of magnitude. This effect was reversible. Consequently, the spot size (i.e., spatial resolution) of the bioluminescence point source imaged through the skin decreased by a factor of 2 (550-nm light) to 3 (650-nm light) after 30 min. Simultaneously, an almost twofold decrease in the amount of light detected by the BLI system was observed, despite the fact that total transmission increased 1.7 times. We have shown here that multiply scattered light is responsible for both observations. We have shown that applying a hyperosmotic clearing agent to the skin of small rodents has the potential to improve spatial resolution of BLI owing to a reduction in the reduced scattering coefficient in the skin by one order of magnitude. However, reducing the scattering coefficient reduces the amount of light reaching the camera due to a reduction in the amount of multiply scattered light that reaches the camera aperture and thus reducing the sensitivity of the method.
0 Communities
1 Members
0 Resources
12 MeSH Terms
Assessing laser-tissue damage with bioluminescent imaging.
Wilmink GJ, Opalenik SR, Beckham JT, Davidson JM, Jansen ED
(2006) J Biomed Opt 11: 041114
MeSH Terms: Animals, Biomimetic Materials, Cells, Cultured, Dose-Response Relationship, Radiation, HSP70 Heat-Shock Proteins, Humans, Lasers, Luminescent Measurements, Mice, Microscopy, Fluorescence, Radiation Dosage, Risk Assessment, Risk Factors, Skin, Skin, Artificial
Show Abstract · Added December 10, 2013
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 and eosin stains verified the presence of the thermally denatured tissue regions. Immunohistochemical analyses confirmed that maximal hsp70 expression occurred at a depth of 150 microm. Bioluminescent microscopy was employed to corroborate these findings. These results indicate that quantitative BLI in engineered tissue equivalents provides a powerful model that enables sequential gene expression studies. Such a model can be used as a high throughput screening platform for laser-tissue interaction studies.
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15 MeSH Terms
In vivo monitoring of pancreatic beta-cells in a transgenic mouse model.
Smith SJ, Zhang H, Clermont AO, Powers AC, Kaufman DB, Purchio AF, West DB
(2006) Mol Imaging 5: 65-75
MeSH Terms: Animals, Diabetes Mellitus, Experimental, Female, Genes, Reporter, Insulin, Insulin Secretion, Insulin-Secreting Cells, Luciferases, Luminescent Measurements, Male, Mice, Mice, Transgenic, Nuclear Proteins, Obesity, Promoter Regions, Genetic, Ribosomal Proteins
Show Abstract · Added August 13, 2010
We generated a transgenic mouse model (RIP-luc) for the in vivo monitoring of pancreatic islet mass and function in response to metabolic disease. Using the rat insulin promoter fused to firefly luciferase, and noninvasive technology to detect luciferase activity, we tracked changes in reporter signal during metabolic disease states and correlated the changes in luciferase signal with metabolic status of the mouse. Transgene expression was found to be specific to the pancreatic islets in this transgenic model. Basal transgene expression was tracked in male and female mice fed either a chow or a high-fat diet and in response to treatment with streptozotocin. Pancreatic bioluminescent signal increased in mice fed a high-fat diet compared with chow-fed animals. In a model of chemically induced diabetes, the bioluminescent signal decreased in accordance with the onset of diabetes and reduction of islet beta-cell number. Preliminary studies using islets transplanted from this transgenic model suggest that in vivo image analysis can also be used to monitor transplanted islet viability and survival in the host. This transgenic model is a useful tool for in vivo studies of pancreatic beta-cells and as a donor for islet transplantation studies.
1 Communities
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0 Resources
16 MeSH Terms