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In JoVE (1)
- Сегментация и измерение объемов жира в мышиной модели ожирения Использование рентгеновской компьютерной томографии
Other Publications (16)
- Bioconjugate Chemistry
- Proceedings of the National Academy of Sciences of the United States of America
- Organic & Biomolecular Chemistry
- Analytical Chemistry
- Molecular Imaging
- Proceedings of the National Academy of Sciences of the United States of America
- Journal of the American Chemical Society
- Angewandte Chemie (International Ed. in English)
- The Journal of Biological Chemistry
- Bioconjugate Chemistry
- Molecular Imaging
- Nature Medicine
- Biotechnology Progress
- Methods in Molecular Biology (Clifton, N.J.)
- Molecular Pharmaceutics
- Chemical Communications (Cambridge, England)
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Articles by Seth T. Gammon in JoVE
Сегментация и измерение объемов жира в мышиной модели ожирения Использование рентгеновской компьютерной томографии
Todd A. Sasser1, Sarah E. Chapman2, Shengting Li1, Caroline Hudson2, Sean P. Orton1, Justin M. Diener3, Seth T. Gammon1, Carlos Correcher4, W. Matthew Leevy2
1Carestream Molecular Imaging, 2Department of Chemistry and Biochemistry, University of Notre Dame, 3Freimann Life Science Center, University of Notre Dame, 4Research and Development, Oncovision, GEM-Imaging S.A.
Жир контент-анализ регулярно проводится в исследованиях с использованием мышиной модели ожирения. Новые методы в маленькое животное КТ и анализ обеспечения продольной подробно богатых жиром контент-анализ. Здесь мы подробно шаг за шагом процедуры для выполнения мелких животных КТ, анализа и визуализации.
Other articles by Seth T. Gammon on PubMed
Quantitative Analysis of Permeation Peptide Complexes Labeled with Technetium-99m: Chiral and Sequence-specific Effects on Net Cell Uptake
Bioconjugate Chemistry. Mar-Apr, 2003 | Pubmed ID: 12643747
This study investigated sequence-specific cell uptake characteristics of Tat basic domain and related permeation peptides with an emphasis on residue chirality, length, and modified side chains. Effects on cell permeation of defined basic domain sequences within a library of 42 different peptides were evaluated using transport of radiolabeled peptides into human Jurkat leukemia cells. All other factors being equal, when the chirality of the peptide sequence was changed from l to d, uptake values increased up to 13-fold. Control experiments showed that the quantitative difference in uptake could not be attributed to increased decomposition of an l- versus a d-peptide by cellular or serum proteases. Furthermore, length, sequence, and type of chelation domain impacted peptide uptake into cells. The highest level of uptake was found with the following peptides: (23) d-Tat-Orn [Ac-rkkrr-orn-rrr-AHA-kgc-amide] and (33) d-poly-Arg(9) [Ac-rrrrrrrrr-AHA-kgc-amide]. The best of these peptide sequences could be employed as in vivo imaging and drug delivery agents to translocate substrates into cells.
Kinetics of Regulated Protein-protein Interactions Revealed with Firefly Luciferase Complementation Imaging in Cells and Living Animals
Proceedings of the National Academy of Sciences of the United States of America. Aug, 2004 | Pubmed ID: 15284440
Signaling pathways regulating proliferation, differentiation, and apoptosis are commonly mediated through protein-protein interactions as well as reversible phosphorylation of proteins. To facilitate the study of regulated protein-protein interactions in cells and living animals, we optimized firefly luciferase protein fragment complementation by screening incremental truncation libraries of N- and C-terminal fragments of luciferase. Fused to the rapamycin-binding domain (FRB) of the kinase mammalian target of rapamycin and FK506-binding protein 12 (FKBP), respectively, the optimized FRB-N-terminal luciferase fragment (NLuc)/C-terminal luciferase fragment (CLuc)-FKBP luciferase complementation imaging (LCI) pair reconstituted luciferase activity in cells upon single-site binding of rapamycin in an FK506-competitive manner. LCI was used in three independent applications. In mice bearing implants of cells expressing the FRB-NLuc/CLuc-FKBP LCI pair, dose- and time-dependent luciferase activity allowed target-specific pharmacodynamic analysis of rapamycin-induced protein-protein interactions in vivo. In cells expressing a Cdc25C-NLuc/CLuc-14-3-3epsilon LCI pair, drug-mediated disruption of cell cycle regulated protein-protein interactions was demonstrated with the protein kinase inhibitor UCN-01 in a phosphoserine-dependent manner. When applied to IFN-gamma-dependent activation of Janus kinase/signal transducer and activator of transcription 1 (STAT1), LCI revealed, in the absence of ligand-induced phosphorylation, STAT1 proteins existing in live cells as preformed dimers. Thus, optimized LCI provides a platform for near real-time detection and characterization of regulated and small molecule-induced protein-protein interactions in intact cells and living animals and should enable a wide range of novel applications in drug discovery, chemical genetics, and proteomics research.
Structure-activity Relationships, Kinetics, Selectivity, and Mechanistic Studies of Synthetic Hydraphile Channels in Bacterial and Mammalian Cells
Organic & Biomolecular Chemistry. Oct, 2005 | Pubmed ID: 16172693
Hydraphile compounds are shown to be cytotoxic to Gram-negative and Gram-positive bacteria, yeast, and mammalian cells. Their cellular toxicity compares favorably with other synthetic ionophores and rivals that potency of natural antibiotics. The effects of structural variations on toxicity are described. The effects of these variations correlate well with previous studies of ion transport in liposomes. Whole cell patch clamping with mammalian cells confirms a channel mechanism in living cells suggesting that this family may comprise novel and flexible pharmacological agents.
Analytical Chemistry. Mar, 2006 | Pubmed ID: 16503603
A wide variety of bioluminescent luciferase proteins are available for use in transcriptional or biochemical reporter assays. However, spectral overlap normally prevents them from being monitored simultaneously. To address this problem, a Java plug-in for ImageJ was written to deconvolute bioluminescent images composed of signals from multiple luciferases. The methodology was validated by testing the program with both simulated and real luciferase images. Bioluminescent images were acquired using a CCD camera equipped with optical filters, and the images were deconvoluted using the ImageJ plug-in. HeLa cells were transfected with either click beetle red luciferase (CBR), click beetle green luciferase (CBG99), or Renilla luciferase (Rluc), and mixed lysates were imaged in varying proportions in a 96-well plate to biochemically validate the methodology. After spectral deconvolution, the predicted, pure luciferase signals could be recovered with maximal cross-talk errors of +/-1.5%. In addition, live cells expressing CBR, CBG99, and Rluc were simultaneously imaged and deconvoluted in 96-well plates to demonstrate the feasibility of applying this methodology to high-throughput applications. Finally, multicolor transcriptional and posttranslational modification reporters were simultaneously imaged and shown to deconvolute normalized IkappaBeta kinase activity in longitudinal assays. Thus, our software provided a rapid, simple, and accurate method for simultaneously measuring multiple bioluminescent reporters in living cells.
Molecular Imaging. Jan-Mar, 2006 | Pubmed ID: 16779965
Rapid and efficient delivery of imaging probes to the cell interior using permeation peptides has enabled novel applications in molecular imaging. Membrane permeant peptides based on the HIV-1 Tat basic domain sequence, GRKKRRQRRR, labeled with fluorophores and fluorescent proteins for optical imaging or with appropriate peptide-based motifs or macrocycles to chelate metals, such as technetium for nuclear scintigraphy and gadolinium for magnetic resonance imaging, have been synthesized. In addition, iron oxide complexes have been functionalized with the Tat basic domain peptides for magnetic resonance imaging applications. Herein we review current applications of permeation peptides in molecular imaging and factors influencing permeation peptide internalization. These diagnostic agents show concentrative cell accumulation and rapid kinetics and display cytosolic and focal nuclear accumulation in human cells. Combining methods, dual-labeled permeation peptides incorporating fluorescein maleimide and chelated technetium have allowed for both qualitative and quantitative analysis of cellular uptake. Imaging studies in mice following intravenous administration of prototypic diagnostic permeation peptides show rapid whole-body distribution allowing for various molecular imaging applications. Strategies to develop permeation peptides into molecular imaging probes have included incorporation of targeting motifs such as molecular beacons or protease cleavable domains that enable selective retention, activatable fluorescence, or targeted transduction. These novel permeation peptide conjugates maintain rapid translocation across cell membranes into intracellular compartments and have the potential for targeted in vivo applications in molecular imaging and combination therapy.
Linkage Between Cellular Communications, Energy Utilization, and Proliferation in Metastatic Neuroendocrine Cancers
Proceedings of the National Academy of Sciences of the United States of America. Aug, 2006 | Pubmed ID: 16895983
To identify metabolic features that support the aggressive behavior of human neuroendocrine (NE) cancers, we examined metastatic prostate NE tumors and derived prostate NE cancer (PNEC) cell lines from a transgenic mouse model using a combination of magic angle spinning NMR spectroscopy, in silico predictions of biotransformations that observed metabolites may undergo, biochemical tests of these predictions, and electrophysiological/calcium imaging studies. Malignant NE cells undergo excitation and increased proliferation when their GABA(A), glutamate, and/or glycine receptors are stimulated, use glutamate and GABA as substrates for NADH biosynthesis, and produce propylene glycol, a precursor of pyruvate derived from glycine that increases levels of circulating free fatty acids through extra-NE cell effects. Treatment of nude mice containing PNEC tumor xenografts with (i) amiloride, a diuretic that inhibits Abp1, an enzyme involved in NE cell GABA metabolism, (ii) carbidopa, an inhibitor of dopa decarboxylase which functions upstream of Abp1, plus (iii) flumazenil, a benzodiazepine antagonist that binds to GABA(A) receptors, leads to significant reductions in tumor growth. These findings may be generally applicable: GeneChip data sets from 471 human neoplasms revealed that components of GABA metabolic pathways, including ABP1, exhibit statistically significant increases in their expression in NE and non-NE cancers.
Optical Imaging of Bacterial Infection in Living Mice Using a Fluorescent Near-infrared Molecular Probe
Journal of the American Chemical Society. Dec, 2006 | Pubmed ID: 17177377
An optical imaging probe was synthesized by attaching a near-infrared carbocyanine fluorophore to an affinity group containing two zinc(II) dipicolylamine (Zn-DPA) units. The probe has a strong and selective affinity for the surfaces of bacteria, and it was used to image infections of Gram-positive S. aureus and Gram-negative E. coli bacteria in living nude mice. After intravenous injection, the probe selectively accumulates at the sites of localized bacterial infections in the thigh muscles of the mice.
Squaraine Rotaxanes: Superior Substitutes for Cy-5 in Molecular Probes for Near-infrared Fluorescence Cell Imaging
Angewandte Chemie (International Ed. in English). 2007 | Pubmed ID: 17585399
The Journal of Biological Chemistry. Mar, 2008 | Pubmed ID: 18203717
In response to a variety of extracellular ligands, nuclear factor-kappaB (NF-kappaB) signaling regulates inflammation, cell proliferation, and apoptosis. It is likely that cells are not continuously exposed to stimulating ligands in vivo but rather experience transient pulses. To study the temporal regulation of NF-kappaB and its major regulator, inhibitor of NF-kappaBalpha (IkappaBalpha), in real time, we utilized a novel transcriptionally coupled IkappaBalpha-firefly luciferase fusion reporter and characterized the dynamics and responsiveness of IkappaBalpha processing upon a short 30-s pulse of tumor necrosis factor alpha (TNFalpha) or a continuous challenge of TNFalpha following a 30-s preconditioning pulse. Strikingly, a 30-s pulse of TNFalpha robustly activated inhibitor of NF-kappaB kinase (IKK), leading to IkappaBalpha degradation, NF-kappaB nuclear translocation, and strong transcriptional up-regulation of IkappaBalpha. Furthermore, we identified a transient refractory period (lasting up to 120 min) following preconditioning, during which the cells were not able to fully degrade IkappaBalpha upon a second TNFalpha challenge. Kinase assays of IKK activity revealed that regulation of IKK activity correlated in part with this transient refractory period. In contrast, experiments involving sequential exposure to TNFalpha and interleukin-1beta indicated that receptor dynamics could not explain this phenomenon. Utilizing a well accepted computational model of NF-kappaB dynamics, we further identified an additional layer of regulation, downstream of IKK, that may govern the temporal capacity of cells to respond to a second proinflammatory insult. Overall, the data suggested that nuclear export of NF-kappaB.IkappaBalpha complexes represented another rate-limiting step that may impact this refractory period, thereby providing an additional regulatory mechanism.
Noninvasive Optical Imaging of Staphylococcus Aureus Bacterial Infection in Living Mice Using a Bis-dipicolylamine-Zinc(II) Affinity Group Conjugated to a Near-infrared Fluorophore
Bioconjugate Chemistry. Mar, 2008 | Pubmed ID: 18260609
Optical imaging of bacterial infection in living animals is usually conducted with genetic reporters such as light-emitting enzymes or fluorescent proteins. However, there are many circumstances where genetic reporters are not applicable, and there is a need for exogenous synthetic probes that can selectively target bacteria. The focus of this study is a fluorescent imaging probe that is composed of a bacterial affinity group conjugated to a near-infrared dye. The affinity group is a synthetic zinc (II) coordination complex that targets the anionic surfaces of bacterial cells. The probe allows detection of Staphylococcus aureus infection (5 x 10 (7) cells) in a mouse leg infection model using whole animal near-infrared fluorescence imaging. Region of interest analysis showed that the signal ratio for infected leg to uninfected leg reaches 3.9 +/- 0.5 at 21 h postinjection of the probe. Ex vivo imaging of the organs produced a signal ratio of 8 for infected to uninfected leg. Immunohistochemical analysis confirmed that the probe targeted the bacterial cells in the infected tissue. Optimization of the imaging filter set lowered the background signal due to autofluorescence and substantially improved imaging contrast. The study shows that near-infrared molecular probes are amenable to noninvasive optical imaging of localized S. aureus infection.
Molecular Imaging. Sep-Oct, 2008 | Pubmed ID: 19123992
Salmonella Typhimurium is a common cause of gastroenteritis in humans and also localizes to neoplastic tumors in animals. Invasion of specific eukaryotic cells is a key mechanism of Salmonella interactions with host tissues. Early stages of gastrointestinal cell invasion are mediated by a Salmonella type III secretion system, powered by the adenosine triphosphatase invC. The aim of this work was to characterize the invC dependence of invasion kinetics into disparate eukaryotic cells traditionally used as models of gut epithelium or neoplasms. Thus, a nondestructive real-time assay was developed to report eukaryotic cell invasion kinetics using lux+ Salmonella that contain chromosomally integrated luxCDABE genes. Bioluminescence-based invasion assays using lux+ Salmonella exhibited inoculum dose-response correlation, distinguished invasion-competent from invasion-incompetent Salmonella, and discriminated relative Salmonella invasiveness in accordance with environmental conditions that induce invasion gene expression. In standard gentamicin protection assays, bioluminescence from lux+ Salmonella correlated with recovery of colony-forming units of internalized bacteria and could be visualized by bioluminescence microscopy. Furthermore, this assay distinguished invasion-competent from invasion-incompetent bacteria independent of gentamicin treatment in real time. Bioluminescence reported Salmonella invasion of disparate eukaryotic cell lines, including neoplastic melanoma, colon adenocarcinoma, and glioma cell lines used in animal models of malignancy. In each case, Salmonella invasion of eukaryotic cells was invC dependent.
Nature Medicine. Apr, 2009 | Pubmed ID: 19305414
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.
Rational Design of Novel Red-shifted BRET Pairs: Platforms for Real-time Single-chain Protease Biosensors
Biotechnology Progress. Mar-Apr, 2009 | Pubmed ID: 19330851
Bioluminescence resonance energy transfer (BRET) systems to date have been dominated by use of blue-green Renilla luciferase (Rluc) as the energy donor. Although effective in many cases, the expense and unfavorable biochemical attributes of the substrate (phenylcoelenterazine) limit utility of Rluc-based BRET systems. Herein we report a series of novel BRET pairs based on luciferases that utilize D-luciferin, resulting in red-shifted photonic outputs, favorable biochemical attributes, and increased efficacy. We developed a modified Förster equation to predict optimal BRET luciferase donor-fluorophore pairs and identified tdTomato as the optimal red fluorophore acceptor for click beetle green luciferase (CBG). A prototypical single-chain protease biosensor, capable of reporting on executioner caspase activity in live cells and in real-time, was generated by inserting a DEVD linker between CBG and tdTomato and validated in vitro with recombinant caspases and in cellulo with apoptosis-sensitive and -resistant cell lines. High signal-to-noise ratios ( approximately 33) and Z' factors (0.85) were observed in live cell longitudinal studies, sufficient for high-throughput screening. Thus, we illustrate a general methodology for the rational design of new BRET systems and provide a novel single-chain BRET protease biosensor that is long lived, red-shifted, and utilizes D-luciferin.
Targeted Chemotherapy in Drug-resistant Tumors, Noninvasive Imaging of P-glycoprotein-mediated Functional Transport in Cancer, and Emerging Role of Pgp in Neurodegenerative Diseases
Methods in Molecular Biology (Clifton, N.J.). 2010 | Pubmed ID: 19949924
Multidrug resistance (MDR) mediated by overexpression of P-glycoprotein (Pgp) is one of the best characterized transporter-mediated barriers to successful chemotherapy in cancer patients and is also a rapidly emerging target in the progression of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Therefore, strategies capable of delivering chemotherapeutic agents into drug-resistant tumors and targeted radiopharmaceuticals acting as ultrasensitive molecular imaging probes for detecting functional Pgp expression in vivo could be expected to play a vital role in systemic biology as personalized medicine gains momentum in the twenty-first century. While targeted therapy could be expected to deliver optimal doses of chemotherapeutic drugs into the desired targets, the interrogation of Pgp-mediated transport activity in vivo via noninvasive imaging techniques (SPECT and PET) would be beneficial in stratification of patient populations likely to benefit from a given therapeutic treatment, thereby assisting management of drug resistance in cancer and treatment of neurodegenerative diseases. Both strategies could play a vital role in advancement of personalized treatments in cancer and neurodegenerative diseases. Via this tutorial, authors make an attempt in outlining these strategies and discuss their strengths and weaknesses.
In Vivo Optical Imaging of Acute Cell Death Using a Near-infrared Fluorescent Zinc-dipicolylamine Probe
Molecular Pharmaceutics. Apr, 2011 | Pubmed ID: 21323375
Cell death is a fundamental biological process that is present in numerous disease pathologies. Fluorescent probes that detect cell death have been developed for a myriad of research applications ranging from microscopy to in vivo imaging. Here we describe a synthetic near-infrared (NIR) conjugate of zinc(II)-dipicolylamine (Zn²+-DPA) for in vivo imaging of cell death. Chemically induced in vivo models of myopathy were established using an ionphore, ethanol, or ketamine as cytotoxins. The Zn²+-DPA fluorescent probe or corresponding control was subsequently injected, and whole animal fluorescence imaging demonstrated probe uptake at the site of muscle damage, which was confirmed by ex vivo and histological analyses. Further, a comparative study with a NIR fluorescent conjugate Annexin V showed less intense uptake at the site of muscle damage and high accumulation in the bladder. The results indicate that the fluorescent Zn²+-DPA conjugate is an effective probe for in vivo cell death detection and in some cases may be an appropriate alternative to fluorescent Annexin V conjugates.
Chemical Communications (Cambridge, England). Jul, 2011 | Pubmed ID: 21681307
Synthetic ion channel hydraphiles, which are known to infiltrate membranes and disrupt ion homeostasis, were tested as direct injection toxins in live mice as potential schlerotic agents. The study uses a near-IR dye to image and evaluate the success of the approach.