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Sample records for quantitative in-vivo functional

  1. Photosensitizer quantitation in vivo by flourescence microsampling

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    Pogue, Brian W.; Burke, Gregory C.; Lee, Claudia C.; Hoopes, P. Jack

    2000-06-01

    Photodynamic therapy can provide a reliable method of tumor destruction when the appropriate dosimetry is applied. Current dosimetry practice involves quantification of the drug and light doses applied to the tumor, but it would be desirable to monitor in vivo light and drug levels to provide the most accurate determination of dosimetry. In vivo measurements can be used to minimize variations in treatment response due to inter-animal variability, by providing animal-specific or patient-specific treatment planning. This study reports on the development of a micro-sampling method to measure fluorescence from tissue, which is not significantly affected by the tissue optical properties. The system measures fluorescence from the surface of a tissue, using a fiber bundle composed of individual 100 micron fibers which ar all spaced apart by 700 microns from one another at the tissue contact end. This design provides sampling of the fluorescence at multiple sites to increase the signal intensity, while maintaining a micro- sampling of the tissue volume just below the surface. The calibration studies here indicate that the 1/e sampling depth is near 60 microns when measured in optical phantoms, which are similar to typical tissue properties. The probe fluorescence signal is independent of blood concentration up to a maximum of 10% blood by volume, which is similar to most tumor tissue. Animal tests indicate that the sensitivity to drug concentration is essentially the same in when measured in murine liver and muscle tissues, both in vivo and ex vivo. These preliminary calibration results suggest that the probe can be used to measure photosensitizer uptake in vivo non- invasively and rapidly via conversion of fluorescence intensity to photosensitizer concentration.

  2. Towards in vivo focal cortical dysplasia phenotyping using quantitative MRI.

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    Adler, Sophie; Lorio, Sara; Jacques, Thomas S; Benova, Barbora; Gunny, Roxana; Cross, J Helen; Baldeweg, Torsten; Carmichael, David W

    2017-01-01

    Focal cortical dysplasias (FCDs) are a range of malformations of cortical development each with specific histopathological features. Conventional radiological assessment of standard structural MRI is useful for the localization of lesions but is unable to accurately predict the histopathological features. Quantitative MRI offers the possibility to probe tissue biophysical properties in vivo and may bridge the gap between radiological assessment and ex-vivo histology. This review will cover histological, genetic and radiological features of FCD following the ILAE classification and will explain how quantitative voxel- and surface-based techniques can characterise these features. We will provide an overview of the quantitative MRI measures available, their link with biophysical properties and finally the potential application of quantitative MRI to the problem of FCD subtyping. Future research linking quantitative MRI to FCD histological properties should improve clinical protocols, allow better characterisation of lesions in vivo and tailored surgical planning to the individual.

  3. Quantitative Susceptibility Mapping-Based Microscopy of Magnetic Resonance Venography (QSM-mMRV for In Vivo Morphologically and Functionally Assessing Cerebromicrovasculature in Rat Stroke Model.

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    Meng-Chi Hsieh

    Full Text Available Abnormal cerebral oxygenation and vessel structure is a crucial feature of stroke. An imaging method with structural and functional information is necessary for diagnosis of stroke. This study applies QSM-mMRV (quantitative susceptibility mapping-based microscopic magnetic resonance venography for noninvasively detecting small cerebral venous vessels in rat stroke model. First, susceptibility mapping is optimized and calculated from magnetic resonance (MR phase images of a rat brain. Subsequently, QSM-mMRV is used to simultaneously provide information on microvascular architecture and venous oxygen saturation (SvO2, both of which can be used to evaluate the physiological and functional characteristics of microvascular changes for longitudinally monitoring and therapeutically evaluating a disease model. Morphologically, the quantification of vessel sizes using QSM-mMRV was 30% smaller than that of susceptibility-weighted imaging (SWI, which eliminated the overestimation of conventional SWI. Functionally, QSM-mMRV estimated an average SvO2 ranging from 73% to 85% for healthy rats. Finally, we also applied QSM to monitor the revascularization of post-stroke vessels from 3 to 10 days after reperfusion. QSM estimations of SvO2 were comparable to those calculated using the pulse oximeter standard metric. We conclude that QSM-mMRV is useful for longitudinally monitoring blood oxygen and might become clinically useful for assessing cerebrovascular diseases.

  4. Quantitative Susceptibility Mapping-Based Microscopy of Magnetic Resonance Venography (QSM-mMRV) for In Vivo Morphologically and Functionally Assessing Cerebromicrovasculature in Rat Stroke Model.

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    Hsieh, Meng-Chi; Tsai, Ching-Yi; Liao, Min-Chiao; Yang, Jenq-Lin; Su, Chia-Hao; Chen, Jyh-Horng

    2016-01-01

    Abnormal cerebral oxygenation and vessel structure is a crucial feature of stroke. An imaging method with structural and functional information is necessary for diagnosis of stroke. This study applies QSM-mMRV (quantitative susceptibility mapping-based microscopic magnetic resonance venography) for noninvasively detecting small cerebral venous vessels in rat stroke model. First, susceptibility mapping is optimized and calculated from magnetic resonance (MR) phase images of a rat brain. Subsequently, QSM-mMRV is used to simultaneously provide information on microvascular architecture and venous oxygen saturation (SvO2), both of which can be used to evaluate the physiological and functional characteristics of microvascular changes for longitudinally monitoring and therapeutically evaluating a disease model. Morphologically, the quantification of vessel sizes using QSM-mMRV was 30% smaller than that of susceptibility-weighted imaging (SWI), which eliminated the overestimation of conventional SWI. Functionally, QSM-mMRV estimated an average SvO2 ranging from 73% to 85% for healthy rats. Finally, we also applied QSM to monitor the revascularization of post-stroke vessels from 3 to 10 days after reperfusion. QSM estimations of SvO2 were comparable to those calculated using the pulse oximeter standard metric. We conclude that QSM-mMRV is useful for longitudinally monitoring blood oxygen and might become clinically useful for assessing cerebrovascular diseases.

  5. In vivo quantitative susceptibility mapping (QSM in Alzheimer's disease.

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    Julio Acosta-Cabronero

    Full Text Available BACKGROUND: This study explores the magnetostatic properties of the Alzheimer's disease brain using a recently proposed, magnetic resonance imaging, postprocessed contrast mechanism. Quantitative susceptibility mapping (QSM has the potential to monitor in vivo iron levels by reconstructing magnetic susceptibility sources from field perturbations. However, with phase data acquired at a single head orientation, the technique relies on several theoretical approximations and requires fast-evolving regularisation strategies. METHODS: In this context, the present study describes a complete methodological framework for magnetic susceptibility measurements with a review of its theoretical foundations. FINDINGS AND SIGNIFICANCE: The regional and whole-brain cross-sectional comparisons between Alzheimer's disease subjects and matched controls indicate that there may be significant magnetic susceptibility differences for deep brain nuclei--particularly the putamen--as well as for posterior grey and white matter regions. The methodology and findings described suggest that the QSM method is ready for larger-scale clinical studies.

  6. In vivo measurement of protein functional changes

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    Aili Wang, Zhicheng Zhang, Qinyi Zhao

    2009-01-01

    Full Text Available Conformational changes in proteins are fundamental to all biological functions. In protein science, the concept of protein flexibility is widely used to describe protein dynamics and thermodynamic properties that control protein conformational changes. In this study, we show that urea, which has strong sedative potency, can be administered to fish at high concentrations, and that protein functional changes related to anesthesia induction can be measured in vivo. Ctenopharyngodon idellus (the grass carp has two different types of N-methyl d-aspartate (NMDA receptors, urea-insensitive and urea-sensitive, which are responsible for the heat endurance of fish. The urea-sensitive NMDA receptor showed high protein flexibility, the gamma aminobutyric acid (GABA receptor showed less flexibility, and the protein that is responsible for ethanol anesthesia showed the lowest flexibility. The results suggest that an increase in protein flexibility underlies the fundamental biophysical mechanisms of volatile general anesthetics.

  7. Quantitative analysis of in vivo confocal microscopy images: a review.

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    Patel, Dipika V; McGhee, Charles N

    2013-01-01

    In vivo confocal microscopy (IVCM) is a non-invasive method of examining the living human cornea. The recent trend towards quantitative studies using IVCM has led to the development of a variety of methods for quantifying image parameters. When selecting IVCM images for quantitative analysis, it is important to be consistent regarding the location, depth, and quality of images. All images should be de-identified, randomized, and calibrated prior to analysis. Numerous image analysis software are available, each with their own advantages and disadvantages. Criteria for analyzing corneal epithelium, sub-basal nerves, keratocytes, endothelium, and immune/inflammatory cells have been developed, although there is inconsistency among research groups regarding parameter definition. The quantification of stromal nerve parameters, however, remains a challenge. Most studies report lower inter-observer repeatability compared with intra-observer repeatability, and observer experience is known to be an important factor. Standardization of IVCM image analysis through the use of a reading center would be crucial for any future large, multi-centre clinical trials using IVCM.

  8. Novel in vivo techniques to visualize kidney anatomy and function.

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    Peti-Peterdi, János; Kidokoro, Kengo; Riquier-Brison, Anne

    2015-07-01

    Intravital imaging using multiphoton microscopy (MPM) has become an increasingly popular and widely used experimental technique in kidney research over the past few years. MPM allows deep optical sectioning of the intact, living kidney tissue with submicron resolution, which is unparalleled among intravital imaging approaches. MPM has solved a long-standing critical technical barrier in renal research to study several complex and inaccessible cell types and anatomical structures in vivo in their native environment. Comprehensive and quantitative kidney structure and function MPM studies helped our better understanding of the cellular and molecular mechanisms of the healthy and diseased kidney. This review summarizes recent in vivo MPM studies with a focus on the glomerulus and the filtration barrier, although select, glomerulus-related renal vascular and tubular functions are also mentioned. The latest applications of serial MPM of the same glomerulus in vivo, in the intact kidney over several days, during the progression of glomerular disease are discussed. This visual approach, in combination with genetically encoded fluorescent markers of cell lineage, has helped track the fate and function (e.g., cell calcium changes) of single podocytes during the development of glomerular pathologies, and provided visual proof for the highly dynamic, rather than static, nature of the glomerular environment. Future intravital imaging applications have the promise to further push the limits of optical microscopy, and to advance our understanding of the mechanisms of kidney injury. Also, MPM will help to study new mechanisms of tissue repair and regeneration, a cutting-edge area of kidney research.

  9. Photoacoustic molecular imaging for in vivo liver iron quantitation

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    Maccarinelli, Federica; Carmona, Fernando; Regoni, Maria; Arosio, Paolo

    2016-05-01

    A recent study showed that ferritin is a suitable endogenous contrast agent for photoacoustic molecular imaging in cultured mammalian cells. We have therefore tested whether this imaging technique can be used for in vivo quantification of iron in mouse livers. To verify this hypothesis, we used multispectral optoacoustic tomography (MSOT) to image albino CD1 mice before and after experimental iron loading. Postmortem assays showed that the iron treatment caused a 15-fold increase in liver iron and a 40-fold increase in liver ferritin levels, while in vivo longitudinal analysis using MSOT revealed just a 1.6-fold increase in the ferritin/iron photoacoustic signal in the same animals. We conclude that MSOT can monitor changes in ferritin/iron levels in vivo, but its sensitivity is much lower than that of ex vivo iron assays.

  10. Genetic analysis of basophil function in vivo

    OpenAIRE

    Sullivan, Brandon M.; Liang, Hong-Erh; Bando, Jennifer K.; Wu, Davina; Cheng, Laurence E.; McKerrow, James K.; Allen, Christopher D.C.; Locksley, Richard M.

    2011-01-01

    Contributions by basophils to allergic and helminth immunity remain incompletely defined. Using sensitive IL-4 reporter alleles, we demonstrate that basophil IL-4 production occurs by a CD4+ T cell-dependent process restricted to affected peripheral tissues. We genetically marked and specifically deleted basophils and demonstrate that basophils do not mediate TH2 priming in vivo. Two-photon imaging confirmed that basophils do not interact with antigen-specific T cells in lymph nodes, but can ...

  11. Genetic analysis of basophil function in vivo.

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    Sullivan, Brandon M; Liang, Hong-Erh; Bando, Jennifer K; Wu, Davina; Cheng, Laurence E; McKerrow, James K; Allen, Christopher D C; Locksley, Richard M

    2011-06-01

    Contributions by basophils to allergic and helminth immunity remain incompletely defined. Using sensitive interleukin 4 (Il4) reporter alleles, we demonstrate here that basophil IL-4 production occurs by a CD4(+) T cell-dependent process restricted to the peripheral tissues affected. We genetically marked and achieved specific deletion of basophils and found that basophils did not mediate T helper type 2 (T(H)2) priming in vivo. Two-photon imaging confirmed that basophils did not interact with antigen-specific T cells in lymph nodes but engaged in prolonged serial interactions with T cells in lung tissues. Although targeted deletion of IL-4 and IL-13 in either CD4(+) T cells or basophils had a minimal effect on worm clearance, deletion from both lineages demonstrated a nonredundant role for basophil cytokines in primary helminth immunity.

  12. A practical and sensitive method of quantitating lymphangiogenesis in vivo.

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    Majumder, Mousumi; Xin, Xiping; Lala, Peeyush K

    2013-07-01

    To address the inadequacy of current assays, we developed a directed in vivo lymphangiogenesis assay (DIVLA) by modifying an established directed in vivo angiogenesis assay. Silicon tubes (angioreactors) were implanted in the dorsal flanks of nude mice. Tubes contained either growth factor-reduced basement membrane extract (BME)-alone (negative control) or BME-containing vascular endothelial growth factor (VEGF)-D (positive control for lymphangiogenesis) or FGF-2/VEGF-A (positive control for angiogenesis) or a high VEGF-D-expressing breast cancer cell line MDA-MD-468LN (468-LN), or VEGF-D-silenced 468LN. Lymphangiogenesis was detected superficially with Evans Blue dye tracing and measured in the cellular contents of angioreactors by multiple approaches: lymphatic vessel endothelial hyaluronan receptor-1 (Lyve1) protein (immunofluorescence) and mRNA (qPCR) expression and a visual scoring of lymphatic vs blood capillaries with dual Lyve1 (or PROX-11 or Podoplanin)/Cd31 immunostaining in cryosections. Lymphangiogenesis was absent with BME, high with VEGF-D or VEGF-D-producing 468LN cells and low with VEGF-D-silenced 468LN. Angiogenesis was absent with BME, high with FGF-2/VEGF-A, moderate with 468LN or VEGF-D and low with VEGF-D-silenced 468LN. The method was reproduced in a syngeneic murine C3L5 tumor model in C3H/HeJ mice with dual Lyve1/Cd31 immunostaining. Thus, DIVLA presents a practical and sensitive assay of lymphangiogenesis, validated with multiple approaches and markers. It is highly suited to identifying pro- and anti-lymphangiogenic agents, as well as shared or distinct mechanisms regulating lymphangiogenesis vs angiogenesis, and is widely applicable to research in vascular/tumor biology.

  13. A quantitative model for the in vivo assessment of drug binding sites with positron emission tomography

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    Mintun, M.A.; Raichle, M.E.; Kilbourn, M.R.; Wooten, G.F.; Welch, M.J.

    1984-03-01

    We propose an in vivo method for use with positron emission tomography (PET) that results in a quantitative characterization of neuroleptic binding sites using radiolabeled spiperone. The data are analyzed using a mathematical model that describes transport, nonspecific binding, and specific binding in the brain. The model demonstrates that the receptor quantities Bmax (i.e., the number of binding sites) and KD-1 (i.e., the binding affinity) are not separably ascertainable with tracer methodology in human subjects. We have, therefore, introduced a new term, the binding potential, equivalent to the product BmaxKD-1, which reflects the capacity of a given tissue, or region of a tissue, for ligand-binding site interaction. The procedure for obtaining these measurements is illustrated with data from sequential PET scans of baboons after intravenous injection of carrier-added (18F)spiperone. From these data we estimate the brain tissue nonspecific binding of spiperone to be in the range of 94.2 to 95.3%, and the regional brain spiperone permeability (measured as the permeability-surface area product) to be in the range of 0.025 to 0.036 cm3/(s X ml). The binding potential of the striatum ranged from 17.4 to 21.6; these in vivo estimates compare favorably to in vitro values in the literature. To our knowledge this represents the first direct evidence that PET can be used to characterize quantitatively, locally and in vivo, drug binding sites in brain. The ability to make such measurements with PET should permit the detailed investigation of diseases thought to result from disorders of receptor function.

  14. Improved estimation of the temporal decay function of in vivo metabolite signals

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    Van Ormondt, D.; De Beer, R.; Van der Veen, J.W.C.; Sima, D.M.; Graveron-Demilly, D.

    2015-01-01

    MRI-scanners enable non-invasive, in vivo quantitation of metabolites in, e.g., the brain of a patient. Among other things, this requires adequate estimation of the unknown temporal decay function of the complex-valued signal emanating from the metabolites. We propose a method to render a current de

  15. In Vivo Quantitation of Local Anesthetic Suppression of Leukocyte Adherence

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    Giddon, D. B.; Lindhe, J.

    1972-01-01

    Using intravital microscopy, topically applied amide-type local anesthetics suppressed the adherence of leukocytes to the venular endothelium within surgical defects in the hamster cheek pouch. The response was reversible with physiologic saline and was localized to venules within the defect. Quantitation in terms of the percent of initially adhering leukocytes remaining in place on the venule wall at each minute following application of lidocaine and physiologic saline, respectively, revealed the suppression to be reliably related to the concentration, viz: 20.0 >10.0 >5.0 >0.0 mg ml of commercially available Xylocaine® (lidocaine) HCl. ImagesFig 1Fig 1 PMID:5049429

  16. Quantitative in vivo magnetic resonance spectroscopy using synthetic signal injection.

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    Kenneth I Marro

    Full Text Available Accurate conversion of magnetic resonance spectra to quantitative units of concentration generally requires compensation for differences in coil loading conditions, the gains of the various receiver amplifiers, and rescaling that occurs during post-processing manipulations. This can be efficiently achieved by injecting a precalibrated, artificial reference signal, or pseudo-signal into the data. We have previously demonstrated, using in vitro measurements, that robust pseudo-signal injection can be accomplished using a second coil, called the injector coil, properly designed and oriented so that it couples inductively with the receive coil used to acquire the data. In this work, we acquired nonlocalized phosphorous magnetic resonance spectroscopy measurements from resting human tibialis anterior muscles and used pseudo-signal injection to calculate the Pi, PCr, and ATP concentrations. We compared these results to parallel estimates of concentrations obtained using the more established phantom replacement method. Our results demonstrate that pseudo-signal injection using inductive coupling provides a robust calibration factor that is immune to coil loading conditions and suitable for use in human measurements. Having benefits in terms of ease of use and quantitative accuracy, this method is feasible for clinical use. The protocol we describe could be readily translated for use in patients with mitochondrial disease, where sensitive assessment of metabolite content could improve diagnosis and treatment.

  17. Rational design of surface/interface chemistry for quantitative in vivo monitoring of brain chemistry.

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    Zhang, Meining; Yu, Ping; Mao, Lanqun

    2012-04-17

    To understand the molecular basis of brain functions, researchers would like to be able to quantitatively monitor the levels of neurochemicals in the extracellular fluid in vivo. However, the chemical and physiological complexity of the central nervous system (CNS) presents challenges for the development of these analytical methods. This Account describes the rational design and careful construction of electrodes and nanoparticles with specific surface/interface chemistry for quantitative in vivo monitoring of brain chemistry. We used the redox nature of neurochemicals at the electrode/electrolyte interface to establish a basis for monitoring specific neurochemicals. Carbon nanotubes provide an electrode/electrolyte interface for the selective oxidation of ascorbate, and we have developed both in vivo voltammetry and an online electrochemical detecting system for continuously monitoring this molecule in the CNS. Although Ca(2+) and Mg(2+) are involved in a number of neurochemical signaling processes, they are still difficult to detect in the CNS. These divalent cations can enhance electrocatalytic oxidation of NADH at an electrode modified with toluidine blue O. We used this property to develop online electrochemical detection systems for simultaneous measurements of Ca(2+) and Mg(2+) and for continuous selective monitoring of Mg(2+) in the CNS. We have also harnessed biological schemes for neurosensing in the brain to design other monitoring systems. By taking advantage of the distinct reaction properties of dopamine (DA), we have developed a nonoxidative mechanism for DA sensing and a system that can potentially be used for continuously sensing of DA release. Using "artificial peroxidase" (Prussian blue) to replace a natural peroxidase (horseradish peroxidase, HRP), our online system can simultaneously detect basal levels of glucose and lactate. By substituting oxidases with dehydrogenases, we have used enzyme-based biosensing schemes to develop a physiologically

  18. In vivo intraoperative hypoglossal nerve stimulation for quantitative tongue motion analysis

    NARCIS (Netherlands)

    Alphen, van M.J.A.; Eskes, M.; Smeele, L.E.; Balm, A.J.M.; Heijden, van der F.

    2015-01-01

    This is the first study quantitatively measuring tongue motion in 3D after in vivo intraoperative neurostimulation of the hypoglossal nerve and its branches during a neck dissection procedure. Firstly, this study is performed to show whether this set-up is suitable for innervating different muscles

  19. Resurrection of DNA function in vivo from an extinct genome.

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    Pask, Andrew J; Behringer, Richard R; Renfree, Marilyn B

    2008-05-21

    There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine), obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

  20. Resurrection of DNA function in vivo from an extinct genome.

    Directory of Open Access Journals (Sweden)

    Andrew J Pask

    Full Text Available There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine, obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

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

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    Chen, C.-C. [Cancer Research Division, National Health Research Institute, Miaoli 350, Taiwan (China); Hwang, Jeng-Jong [Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China)]. E-mail: jjhwang@ym.edu.tw; Ting, G. [Cancer Research Division, National Health Research Institute, Miaoli 350, Taiwan (China); Tseng, Y.-L. [Taiwan Liposome Company, Taipei 115, Taiwan (China); Wang, S.-J. [Department of Nuclear Medicine, Veterans General Hospital, Taipei 112, Taiwan (China); Whang-Peng, J. [Cancer Research Division, National Health Research Institute, Miaoli 350, Taiwan (China)

    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 (R {sup 2}=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{sup 3} (R {sup 2}=0.907). {gamma} Scintigraphy combined with [{sup 131}I]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.

  2. In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue.

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    Alexander Georg Khandoga

    Full Text Available Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii in vivo near-infrared reflected-light oblique transillumination (RLOT microscopy for the visualization of leukocyte motility and morphology, and iii in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3, platelet-activating factor (PAF] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp mice (mice exhibiting green fluorescent protein-labeled monocytes, we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo.

  3. Adrenergic Receptors From Molecular Structure to in vivo function.

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    Hein, L; Kobilka, B K

    1997-07-01

    Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system as well as many endocrine and parenchymal tissues. Although several hundred G-protein-coupled receptors have been identified, adrenergic receptors, along with the visual pigment rhodopsin, have been among the most extensively studied members of this family of receptors. This review focuses on recent advances in understanding the molecular structure, function, and regulation of adrenergic receptors using in vitro systems and integrates recent transgenic animal models that were generated to study the adrenergic system in vivo. (Trends Cardiovasc Med 1997;7:137-145). © 1997, Elsevier Science Inc.

  4. Noninvasive and Quantitative Assessment of In Vivo Fetomaternal Interface Angiogenesis Using RGD-Based Fluorescence

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    M. Keramidas

    2014-01-01

    Full Text Available Angiogenesis is a key process for proper placental development and for the success of pregnancy. Although numerous in vitro methods have been developed for the assessment of this process, relatively few reliable in vivo methods are available to evaluate this activity throughout gestation. Here we report an in vivo technique that specifically measures placental neovascularization. The technique is based on the measurement of a fluorescent alpha v beta 3 (αvβ3 integrin-targeting molecule called Angiolone-Alexa-Fluor 700. The αvβ3 integrin is highly expressed by endothelial cells during the neovascularization and by trophoblast cells during their invasion of the maternal decidua. Angiolone was injected to gravid mice at 6.5 and 11.5 days post coitus (dpc. The fluorescence was analyzed one day later at 7.5 and 12.5 dpc, respectively. We demonstrated that (i Angiolone targets αvβ3 protein in the placenta with a strong specificity, (ii this technique is quantitative as the measurement was correlated to the increase of the placental size observed with increasing gestational age, and (iii information on the outcome is possible, as abnormal placentation could be detected early on during gestation. In conclusion, we report the validation of a new noninvasive and quantitative method to assess the placental angiogenic activity, in vivo.

  5. Measurement of bacterial gene expression in vivo by laser capture microdissection and quantitative real-time RT-PCR

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    Schou, Kirstine Klitgaard; Jensen, Tim Kåre; Angen, Øystein

    2007-01-01

    Due to the relative small number of bacterial pathogens present in an infected host, exploration of pathogen gene expression in vivo is challenging. This study reports the development of a protocol for quantifying bacterial gene expression in vivo in Actinobacillus pleuropneumoniae using laser ca...... capture microdissection and real-time quantitative RT-PCR....

  6. Real-Time, In Vivo Monitoring, and Quantitative Assessment of Intra-Arterial Vasospasm Therapy.

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    Gölitz, Philipp; Kaschka, Iris; Lang, Stefan; Roessler, Karl; Knossalla, Frauke; Doerfler, Arnd

    2016-08-01

    Our study aimed to evaluate whether the effect of an intra-arterial vasospasm therapy can be assessed quantitatively by in vivo blood flow analysis using the postprocessing algorithm parametric color coding (PCC). We evaluated 17 patients presenting with acute clinical deterioration due to vasospasm following subarachnoidal hemorrhage treated with intra-arterial nimodipine application. Pre- and post-interventional DSA series were post-processed by PCC. The relative time to maximum opacification (rTmax) was calculated in 14 arterially and venously located points of interest. From that data, the pre- and post-interventional cerebral circulation time (CirT) was calculated. Additionally, the arterial vessel diameters were measured. Pre- and post-interventional values were compared and tested for significance, respectively. Flow analysis revealed in all arterial vessel segments a non-statistically significant prolongation of rTmax after treatment. The mean CirT was 5.62 s (±1.19 s) pre-interventionally and 5.16 s (±0.81 s) post-interventionally, and the difference turned out as statistically significant (p = 0.039). A significantly increased diameter was measurable in all arterial segments post-interventionally. PCC is a fast applicable imaging technique that allows via real-time and in vivo blood flow analysis a quantitative assessment of the effect of intra-arterial vasospasm therapy. Our results seem to validate in vivo that an intra-arterial nimodipine application induces not only vasodilatation of the larger vessels, but also improves the microcirculatory flow, leading to a shortened cerebral CirT that reaches normal range post-interventionally. Procedural monitoring via PCC offers the option to compare quantitatively different therapy regimes, which allows optimization of existing approaches and implementation of individualized treatment strategies.

  7. The functions of laminins: lessons from in vivo studies

    DEFF Research Database (Denmark)

    Ryan, M C; Christiano, A M; Engvall, E;

    1996-01-01

    This series of three short reviews is an attempt to summarize our current knowledge of the in vivo tests of hypotheses of laminin functions. The structures of the laminins have been thoroughly reviewed recently (P. Ekblom and R. Timpl, in press), and I will not attempt to repeat this information...... here. Instead, I will focus on the recent evidence gathered from gene knock out experiments in mice and from naturally occurring human and mouse gene mutations. The most obvious lesson from the above studies--other than demonstrating the importance of laminins in general--is that the structural...... normal-other than the anchoring complex itself. The pathology observed in the newborn is believed to be due to the frictional trauma of birth, with the expectation that the function of the fetal skin is normal in utero. The Herlitz epidermolysis bullosa phenotype is obvious immediately at birth...

  8. In Vivo Assessment of Elasticity of Child Rib Cortical Bone Using Quantitative Computed Tomography

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    Y. Zhu

    2017-01-01

    Full Text Available Elasticity of the child rib cortical bone is poorly known due to the difficulties in obtaining specimens to perform conventional tests. It was shown on the femoral cortical bone that elasticity is strongly correlated with density for both children and adults through a unique relationship. Thus, it is assumed that the relationships between the elasticity and density of adult rib cortical bones could be expanded to include that of children. This study estimated in vivo the elasticity of the child rib cortical bone using quantitative computed tomography (QCT. Twenty-eight children (from 1 to 18 y.o. were considered. Calibrated QCT images were prescribed for various thoracic pathologies. The Hounsfield units were converted to bone mineral density (BMD. A relationship between the BMD and the elasticity of the rib cortical bone was applied to estimate the elasticity of children’s ribs in vivo. The estimated elasticity increases with growth (7.1 ± 2.5 GPa at 1 y.o. up to 11.6 ± 1.9 GPa at 18 y.o.. This data is in agreement with the few previous values obtained using direct measurements. This methodology paves the way for in vivo assessment of the elasticity of the child cortical bone based on calibrated QCT images.

  9. Quantitative Analysis of Chemotherapeutic Effects in Tumors Using In Vivo Staining and Correlative Histology

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    Heung Kook Choi

    2005-01-01

    Full Text Available Aims: To microscopically analyze the chemotherapeutic response of tumors using in vivo staining based on an annexinV-Cy5.5 probe and independently asses their apoptotic count using quantitative histological analysis. Methods: Lewis Lung Carcinomas cells, that are sensitive (CS-LLC and resistant (CR-LLC to chemotherapy were implanted in nude mice and grown to tumours. Mice were treated with cyclophosphamide and injected with a Cy5.5-annexinV fluorescent probe. In vivo imaging was performed using Fluorescence Molecular Tomography. Subsequently tumours were excised and prepared for histology. The histological tumour sections were stained for apoptosis using a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL assay. A minimum of ten tissue sections were analyzed per tumour for apoptosis quantification by TUNEL staining and corresponding Cy5.5 distribution. Results: We detected higher levels of apoptosis and corresponding higher levels of Cy5.5 fluorescence in the CS-LLC vs. the CR-LLC tumours. The cell count rate on CS-LLC sections over CR-LLC was found to be ∼2 :1 where the corresponding area observed on Cy5.5 distribution measurements revealed a ∼1.7 :1 ratio of CS-LLC over CR-LLC. These observations are consistent with the higher apoptotic index expected from the CS-LLC cell line. Conclusions: Quantitative analysis of histological slices revealed higher fluorescence and higher apoptotic count in the CS-LLC tumour images compared to the CR-LLC tumour images. These observations demonstrate that the annexinV-Cy5.5 probe sensed the chemotherapeutic effect of cyclophospamide and further confirmed in vivo FMT measurements.

  10. Quantitative ultrasound imaging of therapy response in bladder cancer in vivo.

    Science.gov (United States)

    Tran, William T; Sannachi, Lakshmanan; Papanicolau, Naum; Tadayyon, Hadi; Al Mahrouki, Azza; El Kaffas, Ahmed; Gorjizadeh, Alborz; Lee, Justin; Czarnota, Gregory J

    2016-01-01

    Quantitative ultrasound (QUS) was investigated to monitor bladder cancer treatment response in vivo and to evaluate tumor cell death from combined treatments using ultrasound-stimulated microbubbles and radiation therapy. Tumor-bearing mice (n=45), with bladder cancer xenografts (HT- 1376) were exposed to 9 treatment conditions consisting of variable concentrations of ultrasound-stimulated Definity microbubbles [nil, low (1%), high (3%)], combined with single fractionated doses of radiation (0 Gy, 2 Gy, 8 Gy). High frequency (25 MHz) ultrasound was used to collect the raw radiofrequency (RF) data of the backscatter signal from tumors prior to, and 24 hours after treatment in order to obtain QUS parameters. The calculated QUS spectral parameters included the mid-band fit (MBF), and 0-MHz intercept (SI) using a linear regression analysis of the normalized power spectrum. There were maximal increases in QUS parameters following treatments with high concentration microbubbles combined with 8 Gy radiation: (ΔMBF = +6.41 ± 1.40 (±SD) dBr and SI= + 7.01 ± 1.20 (±SD) dBr. Histological data revealed increased cell death, and a reduction in nuclear size with treatments, which was mirrored by changes in quantitative ultrasound parameters. QUS demonstrated markers to detect treatment effects in bladder tumors in vivo.

  11. Rapid and Quantitative Assessment of Cancer Treatment Response Using In Vivo Bioluminescence Imaging

    Directory of Open Access Journals (Sweden)

    Alnawaz Rehemtulla

    2000-01-01

    Full Text Available Current assessment of orthotopic tumor models in animals utilizes survival as the primary therapeutic end point. In vivo bioluminescence imaging (BLI is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating antineoplastic therapies [1 ]. Using human tumor cell lines constitutively expressing luciferase, the kinetics of tumor growth and response to therapy have been assessed in intraperitoneal [2], subcutaneous, and intravascular [3] cancer models. However, use of this approach for evaluating orthotopic tumor models has not been demonstrated. In this report, the ability of BLI to noninvasively quantitate the growth and therapeuticinduced cell kill of orthotopic rat brain tumors derived from 9L gliosarcoma cells genetically engineered to stably express firefly luciferase (9LLuc was investigated. Intracerebral tumor burden was monitored over time by quantitation of photon emission and tumor volume using a cryogenically cooled CCD camera and magnetic resonance imaging (MRI, respectively. There was excellent correlation (r=0.91 between detected photons and tumor volume. A quantitative comparison of tumor cell kill determined from serial MRI volume measurements and BLI photon counts following 1,3-bis(2-chloroethyl-1-nitrosourea (BCNU treatment revealed that both imaging modalities yielded statistically similar cell kill values (P=.951. These results provide direct validation of BLI imaging as a powerful and quantitative tool for the assessment of antineoplastic therapies in living animals.

  12. In vivo hepatocyte MR imaging using lactose functionalized magnetoliposomes.

    Science.gov (United States)

    Ketkar-Atre, Ashwini; Struys, Tom; Dresselaers, Tom; Hodenius, Michael; Mannaerts, Inge; Ni, Yicheng; Lambrichts, Ivo; Van Grunsven, Leo A; De Cuyper, Marcel; Himmelreich, Uwe

    2014-01-01

    The aim of this study was to assess a novel lactose functionalized magnetoliposomes (MLs) as an MR contrast agent to target hepatocytes as well as to evaluate the targeting ability of MLs for in vivo applications. In the present work, 17 nm sized iron oxide cores functionalized with anionic MLs bearing lactose moieties were used for targeting the asialoglycoprotein receptor (ASGP-r), which is highly expressed in hepatocytes. Non-functionalized anionic MLs were tested as negative controls. The size distribution of lactose and anionic MLs was determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). After intravenous administration of both MLs, contrast enhancement in the liver was observed by magnetic resonance imaging (MRI). Label retention was monitored non-invasively by MRI and validated with Prussian blue staining and TEM for up to eight days post MLs administration. Although the MRI signal intensity did not show significant differences between functionalized and non-functionalized particles, iron-specific Prussian blue staining and TEM analysis confirmed the uptake of lactose MLs mainly in hepatocytes. In contrast, non-functionalized anionic MLs were mainly taken up by Kupffer and sinusoidal cells. Target specificity was further confirmed by high-resolution MR imaging of phantoms containing isolated hepatocytes, Kupffer cell (KCs) and hepatic stellate cells (HSCs) fractions. Hypointense signal was observed for hepatocytes isolated from animals which received lactose MLs but not from animals which received anionic MLs. These data demonstrate that galactose-functionalized MLs can be used as a hepatocyte targeting MR contrast agent to potentially aid in the diagnosis of hepatic diseases if the non-specific uptake by KCs is taken into account.

  13. Proton magnetic resonance spectroscopy of normal human brain and glioma:a quantitive in vivo study

    Institute of Scientific and Technical Information of China (English)

    TONG Zhi-yong; YAMAKI Toshiaki; WANG Yun-jie

    2005-01-01

    Background In vivo proton magnetic resonance spectroscopy (MRS) provides a noninvasive method of examining a wide variety of cerebral metabolites in both healthy subjects and patients with various brain diseases.Absolute metabolite concentrations have been determined using external and internal standards with known concentrations.When an external standard is placed beside the head, variations in signal amplitudes due to B1 field inhomogeneity and static field inhomogeneity may occur.Hence an internal standard is preferable.The purpose of this study was to quantitatively analyze the metabolite concentrations in normal adult brains and gliomas by in vivo proton MRS using the fully relaxed water signal as an internal standard.Methods Between January 1998 and October 2001, 28 healthy volunteers and 16 patients with gliomas were examined by in vivo proton MRS.Single-voxel spectra were acquired using the point-resolved spectroscopic pulse sequence with a 1.5 T scanner (TR/TE/Ave=3000 ms/30 ms/64).Results The calculated concentrations of N-acetyl-asparatate (NAA), creatine (Cre), choline (Cho), and water (H2O) in the normal hemispheric white matter were (23.59±2.62) mmol/L, (13.06±1.8) mmol/L, (4.28±0.8) mmol/L, and (47 280.96±5414.85) mmol/L, respectively.The metabolite concentrations were not necessarily uniform in different parts of the brain.The concentrations of NAA and Cre decreased in all gliomas (P<0.001).The ratios of NAA/Cho and NAA/H2O showed a significant difference between the normal brain and gliomas, and also between the high and low grades (P<0.001).Conclusions Quantitative analysis of in vivo proton MR spectra using the fully relaxed water signal as an internal standard is useful.The concentrations of NAA and the ratios of NAA/H2O and NAA/Cho conduce to discriminating between the glioma and normal brain, and also between the low-grade glioma and high-grade glioma.

  14. Urethral function after cystectomy: a canine in vivo experiment.

    Science.gov (United States)

    Hübner, W A; Trigo-Rocha, F; Plas, E G; Tanagho, E A

    1993-01-01

    To study the function of the pelvic floor and the isolated urethra after removal of the bladder, 5 male and 5 female mongrel dogs were used in an acute in vivo experiment. Urethral pressure changes secondary to unilateral stimulation of the pelvic and pudendal nerves were recorded. After baseline data of the intact system were documented, the following procedures were carried out: separation of the urethra from the bladder neck (prostate), nerve-sparing cystectomy (cystoprostatectomy), and cold-knife incision through the entire length of the proximal urethra. Pressure recordings were repeated after each step of surgery. Pudendal nerve stimulation resulted in rapid and large pressure rises in the distal urethra (reaction typical of striated muscle). This response remained unchanged after all three surgical steps. Pelvic nerve stimulation provoked pressure rises within the urethra of a pattern typical of smooth muscle. The findings persisted after separation of the urethra from the bladder neck (prostate) and after cystectomy, but were not observed after urethrotomy. Contractions secondary to pudendal nerve stimulation were inhibited by curare, which did not affect the reaction to pelvic nerve stimulation. Our experiments demonstrate that in the dog the continuity of bladder and urethra is not required for the function of urethral closure mechanisms. The contractile potency of the urethral smooth muscles remains intact after nerve-sparing cystectomy. We believe that problems with the baseline continence of surrogate bladders should mainly be ascribed to a lack of surgical caution in preserving the autonomic nerves of cystectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. An optimized framework for quantitative magnetization transfer imaging of the cervical spinal cord in vivo.

    Science.gov (United States)

    Battiston, Marco; Grussu, Francesco; Ianus, Andrada; Schneider, Torben; Prados, Ferran; Fairney, James; Ourselin, Sebastien; Alexander, Daniel C; Cercignani, Mara; Gandini Wheeler-Kingshott, Claudia A M; Samson, Rebecca S

    2017-09-16

    To develop a framework to fully characterize quantitative magnetization transfer indices in the human cervical cord in vivo within a clinically feasible time. A dedicated spinal cord imaging protocol for quantitative magnetization transfer was developed using a reduced field-of-view approach with echo planar imaging (EPI) readout. Sequence parameters were optimized based in the Cramer-Rao-lower bound. Quantitative model parameters (i.e., bound pool fraction, free and bound pool transverse relaxation times [ T2F, T2B], and forward exchange rate [kFB ]) were estimated implementing a numerical model capable of dealing with the novelties of the sequence adopted. The framework was tested on five healthy subjects. Cramer-Rao-lower bound minimization produces optimal sampling schemes without requiring the establishment of a steady-state MT effect. The proposed framework allows quantitative voxel-wise estimation of model parameters at the resolution typically used for spinal cord imaging (i.e. 0.75 × 0.75 × 5 mm(3) ), with a protocol duration of ∼35 min. Quantitative magnetization transfer parametric maps agree with literature values. Whole-cord mean values are: bound pool fraction = 0.11(±0.01), T2F = 46.5(±1.6) ms, T2B = 11.0(±0.2) µs, and kFB  = 1.95(±0.06) Hz. Protocol optimization has a beneficial effect on reproducibility, especially for T2B and kFB . The framework developed enables robust characterization of spinal cord microstructure in vivo using qMT. Magn Reson Med, 2017. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of

  16. Fiber optic based multiparametric spectroscopy in vivo: Toward a new quantitative tissue vitality index

    Science.gov (United States)

    Kutai-Asis, Hofit; Barbiro-Michaely, Efrat; Deutsch, Assaf; Mayevsky, Avraham

    2006-02-01

    In our previous publication (Mayevsky et al SPIE 5326: 98-105, 2004) we described a multiparametric fiber optic system enabling the evaluation of 4 physiological parameters as indicators of tissue vitality. Since the correlation between the various parameters may differ in various pathophysiological conditions there is a need for an objective quantitative index that will integrate the relative changes measured in real time by the multiparametric monitoring system into a single number-vitality index. Such an approach to calculate tissue vitality index is critical for the possibility to use such an instrument in clinical environments. In the current presentation we are reporting our preliminary results indicating that calculation of an objective tissue vitality index is feasible. We used an intuitive empirical approach based on the comparison between the calculated index by the computer and the subjective evaluation made by an expert in the field of physiological monitoring. We used the in vivo brain of rats as an animal model in our current studies. The rats were exposed to anoxia, ischemia and cortical spreading depression and the responses were recorded in real time. At the end of the monitoring session the results were analyzed and the tissue vitality index was calculated offline. Mitochondrial NADH, tissue blood flow and oxy-hemoglobin were used to calculate the vitality index of the brain in vivo, where each parameter received a different weight, in each experiment type based on their significance. It was found that the mitochondrial NADH response was the main factor affected the calculated vitality index.

  17. In vivo toxicity of nitroaromatics: A comprehensive quantitative structure-activity relationship study.

    Science.gov (United States)

    Gooch, Aminah; Sizochenko, Natalia; Rasulev, Bakhtiyor; Gorb, Leonid; Leszczynski, Jerzy

    2017-02-07

    The toxicity data of 90 nitroaromatic compounds related to their 50% lethal dose concentration for rats (LD50) were analyzed to develop quantitative structure-activity relationship (QSAR) models. Quantum-chemically calculated descriptors together with molecular descriptors generated by DRAGON, PaDEL, and HiT-QSAR software were utilized to build QSAR models. Quality and validity of the models were determined by internal and external validation techniques. The results show that the toxicity of nitroaromatic compounds depends on various factors, such as the number of nitro-groups, the topological state, and the presence of certain structural fragments. The developed models based on the largest (to date) dataset of nitroaromatics in vivo toxicity showed a good predictive ability. The results provide important input that could be applied in a preliminary assessment of nitroaromatic compounds' toxicity to mammals. Environ Toxicol Chem 2017;9999:1-7. © 2017 SETAC.

  18. Low-frequency quantitative ultrasound imaging of cell death in vivo.

    Science.gov (United States)

    Sadeghi-Naini, Ali; Papanicolau, Naum; Falou, Omar; Tadayyon, Hadi; Lee, Justin; Zubovits, Judit; Sadeghian, Alireza; Karshafian, Raffi; Al-Mahrouki, Azza; Giles, Anoja; Kolios, Michael C; Czarnota, Gregory J

    2013-08-01

    , can detect tissue changes associated with cell death in vivo in response to cancer treatments.

  19. Low-frequency quantitative ultrasound imaging of cell death in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Sadeghi-Naini, Ali; Falou, Omar; Czarnota, Gregory J. [Imaging Research – Physical Science, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5 (Canada); Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5 (Canada); Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario M4N 3M5 (Canada); Department of Radiation Oncology, Faculty of Medicine, University of Toronto, Toronto, Ontario M4N 3M5 (Canada); Papanicolau, Naum; Tadayyon, Hadi [Imaging Research – Physical Science, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5, Canada and Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario M4N 3M5 (Canada); Lee, Justin [Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5, Canada and Department of Radiation Oncology, Faculty of Medicine, University of Toronto, Toronto, Ontario M4N 3M5 (Canada); Zubovits, Judit [Department of Pathology, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5 (Canada); Sadeghian, Alireza [Department of Computer Science, Ryerson University, Toronto, Ontario M5B 2K3 (Canada); Karshafian, Raffi [Department of Physics, Ryerson University, Toronto, Ontario M5B 2K3 (Canada); Al-Mahrouki, Azza; Giles, Anoja [Imaging Research – Physical Science, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5, Canada and Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5 (Canada); Kolios, Michael C. [Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario M4N 3M5, Canada and Department of Physics, Ryerson University, Toronto, Ontario M5B 2K3 (Canada)

    2013-08-15

    , in addition to high-frequency ultrasound, can detect tissue changes associated with cell death in vivo in response to cancer treatments.

  20. Behavior of Endogenous Tumor-Associated Macrophages Assessed In Vivo Using a Functionalized Nanoparticle

    Directory of Open Access Journals (Sweden)

    Antoine Leimgruber

    2009-05-01

    Full Text Available Tumor-associated macrophages (TAMs invade the tumor stroma in many cancers, yet their role is incompletely understood. To visualize and better understand these critical cells in tumor progression, we screened a portfolio of rationally selected, injectable agents to image endogenous TAMs ubiquitously in three different cancer models (colon carcinoma, lung adenocarcinoma, and soft tissue sarcoma. AMTA680, a functionally derivatized magneto-fluorescent nanoparticle, labeled a subset of myeloid cells with an “M2” macrophage phenotype, whereas other neighboring cells, including tumor cells and a variety of other leukocytes, remained unlabeled. We further show that AMTA680-labeled endogenous TAMs are not altered and can be tracked noninvasively at different resolutions and using various imaging modalities, e.g., fluorescence molecular tomography, magnetic resonance imaging, and multiphoton and confocal intravital microscopy. Quantitative assessment of TAM distribution and activity in vivo identified that these cells cluster in delimited foci within tumors, show relatively low motility, and extend cytoplasmic protrusions for prolonged physical interactions with neighboring tumor cells. Noninvasive imaging can also be used to monitor TAM-depleting regimen quantitatively. Thus, AMTA680 or related cell-targeting agents represent appropriate injectable vehicles for in vivo analysis of the tumor microenvironment.

  1. Quantitative Magnetic Particle Imaging Monitors the Transplantation, Biodistribution, and Clearance of Stem Cells In Vivo.

    Science.gov (United States)

    Zheng, Bo; von See, Marc P; Yu, Elaine; Gunel, Beliz; Lu, Kuan; Vazin, Tandis; Schaffer, David V; Goodwill, Patrick W; Conolly, Steven M

    2016-01-01

    Stem cell therapies have enormous potential for treating many debilitating diseases, including heart failure, stroke and traumatic brain injury. For maximal efficacy, these therapies require targeted cell delivery to specific tissues followed by successful cell engraftment. However, targeted delivery remains an open challenge. As one example, it is common for intravenous deliveries of mesenchymal stem cells (MSCs) to become entrapped in lung microvasculature instead of the target tissue. Hence, a robust, quantitative imaging method would be essential for developing efficacious cell therapies. Here we show that Magnetic Particle Imaging (MPI), a novel technique that directly images iron-oxide nanoparticle-tagged cells, can longitudinally monitor and quantify MSC administration in vivo. MPI offers near-ideal image contrast, depth penetration, and robustness; these properties make MPI both ultra-sensitive and linearly quantitative. Here, we imaged, for the first time, the dynamic trafficking of intravenous MSC administrations using MPI. Our results indicate that labeled MSC injections are immediately entrapped in lung tissue and then clear to the liver within one day, whereas standard iron oxide particle (Resovist) injections are immediately taken up by liver and spleen. Longitudinal MPI-CT imaging also indicated a clearance half-life of MSC iron oxide labels in the liver at 4.6 days. Finally, our ex vivo MPI biodistribution measurements of iron in liver, spleen, heart, and lungs after injection showed excellent agreement (R(2) = 0.943) with measurements from induction coupled plasma spectrometry. These results demonstrate that MPI offers strong utility for noninvasively imaging and quantifying the systemic distribution of cell therapies and other therapeutic agents.

  2. A simple, quantitative method using alginate gel to determine rat colonic tumor volume in vivo.

    Science.gov (United States)

    Irving, Amy A; Young, Lindsay B; Pleiman, Jennifer K; Konrath, Michael J; Marzella, Blake; Nonte, Michael; Cacciatore, Justin; Ford, Madeline R; Clipson, Linda; Amos-Landgraf, James M; Dove, William F

    2014-04-01

    Many studies of the response of colonic tumors to therapeutics use tumor multiplicity as the endpoint to determine the effectiveness of the agent. These studies can be greatly enhanced by accurate measurements of tumor volume. Here we present a quantitative method to easily and accurately determine colonic tumor volume. This approach uses a biocompatible alginate to create a negative mold of a tumor-bearing colon; this mold is then used to make positive casts of dental stone that replicate the shape of each original tumor. The weight of the dental stone cast correlates highly with the weight of the dissected tumors. After refinement of the technique, overall error in tumor volume was 16.9% ± 7.9% and includes error from both the alginate and dental stone procedures. Because this technique is limited to molding of tumors in the colon, we utilized the Apc(Pirc/+) rat, which has a propensity for developing colonic tumors that reflect the location of the majority of human intestinal tumors. We have successfully used the described method to determine tumor volumes ranging from 4 to 196 mm³. Alginate molding combined with dental stone casting is a facile method for determining tumor volume in vivo without costly equipment or knowledge of analytic software. This broadly accessible method creates the opportunity to objectively study colonic tumors over time in living animals in conjunction with other experiments and without transferring animals from the facility where they are maintained.

  3. In vivo characterization of regenerative peripheral nerve interface function

    Science.gov (United States)

    Ursu, Daniel C.; Urbanchek, Melanie G.; Nedic, Andrej; Cederna, Paul S.; Gillespie, R. Brent

    2016-04-01

    Objective. Regenerative peripheral nerve interfaces (RPNIs) are neurotized free autologous muscle grafts equipped with electrodes to record myoelectric signals for prosthesis control. Viability of rat RPNI constructs have been demonstrated using evoked responses. In vivo RPNI characterization is the next critical step for assessment as a control modality for prosthetic devices. Approach. Two RPNIs were created in each of two rats by grafting portions of free muscle to the ends of divided peripheral nerves (peroneal in the left and tibial in the right hind limb) and placing bipolar electrodes on the graft surface. After four months, we examined in vivo electromyographic signal activity and compared these signals to muscular electromyographic signals recorded from autologous muscles in two rats serving as controls. An additional group of two rats in which the autologous muscles were denervated served to quantify cross-talk in the electrode recordings. Recordings were made while rats walked on a treadmill and a motion capture system tracked the hind limbs. Amplitude and periodicity of signals relative to gait were quantified, correlation between electromyographic and motion recording were assessed, and a decoder was trained to predict joint motion. Main Results. Raw RPNI signals were active during walking, with amplitudes of 1 mVPP, and quiet during standing, with amplitudes less than 0.1 mVPP. RPNI signals were periodic and entrained with gait. A decoder predicted bilateral ankle motion with greater than 80% reliability. Control group signal activity agreed with literature. Denervated group signals remained quiescent throughout all evaluations. Significance. In vivo myoelectric RPNI activity encodes neural activation patterns associated with gait. Signal contamination from muscles adjacent to the RPNI is minimal, as demonstrated by the low amplitude signals obtained from the Denervated group. The periodicity and entrainment to gait of RPNI recordings suggests the

  4. In vivo Monitoring of Serotonin by Nanomaterial Functionalized Acupuncture Needle

    Science.gov (United States)

    Li, Yu-Tao; Tang, Li-Na; Ning, Yong; Shu, Qing; Liang, Feng-Xia; Wang, Hua; Zhang, Guo-Jun

    2016-06-01

    Acupuncture treatment is amazing but controversial. Up to now, the mechanism of treating diseases by acupuncture and moxibustion is still unclear, especially the occurrence of the molecular events in local acupoints. Herein, we report an extremely stable microsensor by modifying carbon nanotube (CNT) to the tip surface of acupuncture needle and applying this CNT-modified acupuncture needle for real time monitoring of serotonin (5-HT) in vivo. To stabilize CNT modification on the needle tip surface, poly(3,4-ethylenedioxythiophene)(PEDOT) was employed as glue water to stick CNT on the needle. The detection limit of the CNT-modified needle was found to be approximately 50 nM and 78 nM in the PBS and the cell medium, respectively. In addition, the needle showed good selectivity to some inflammatory mediators and some electroactive molecules. For the first time, the CNT-modified needle could be directly probed into rat body for real time monitoring of 5-HT in vivo, showing a great potential for better understanding the mechanism of acupuncture treatment.

  5. Quantitative Detection Method of Hydroxyapatite Nanoparticles Based on Eu(3+) Fluorescent Labeling in Vitro and in Vivo.

    Science.gov (United States)

    Xie, Yunfei; Perera, Thalagalage Shalika Harshani; Li, Fang; Han, Yingchao; Yin, Meizhen

    2015-11-04

    One major challenge for application of hydroxyapatite nanoparticles (nHAP) in nanomedicine is the quantitative detection method. Herein, we exploited one quantitative detection method for nHAP based on the Eu(3+) fluorescent labeling via a simple chemical coprecipitation method. The trace amount of nHAP in cells and tissues can be quantitatively detected on the basis of the fluorescent quantitative determination of Eu(3+) ions in nHAP crystal lattice. The lowest concentration of Eu(3+) ions that can be quantitatively detected is 0.5 nM using DELFIA enhancement solution. This methodology can be broadly applicable for studying the tissue distribution and metabolization of nHAP in vivo.

  6. Renal transplant immunosuppression impairs natural killer cell function in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Olivier Morteau

    Full Text Available Despite an increasing awareness of the importance of innate immunity, the roles of natural killer (NK cells in transplant rejection and antiviral and cancer immunity during immunosuppression have not been clearly defined.To address this issue we have developed a quantitative assay of NK cell function that can be used on clinical samples and have studied the influence of immunosuppression on NK cell function. NK cell degranulation and intracellular interferon (IFN-γ production were determined by flow cytometry of peripheral blood samples.Overnight ex vivo treatment of peripheral blood cells from healthy controls with ciclosporin or tacrolimus inhibited NK cell degranulation and IFN-γ production in a dose-dependent manner. A similar impairment of function was seen in NK cells from patients treated in vivo with calcineurin inhibitors. In the early post-transplant period, there was a variable reduction of NK cell counts after treatment with alemtuzumab and basiliximab.The functional inhibition of NK cells in early transplant patients coincides with the period of maximum susceptibility to viral infections. The ability to assay NK cell function in clinical samples allows assessment of the impact of immunosuppression on these effector cells. This information may be helpful in guiding the titration of immunosuppression in the clinical setting.

  7. In vitro gene regulatory networks predict in vivo function of liver

    Directory of Open Access Journals (Sweden)

    Ang Choo Y

    2010-11-01

    Full Text Available Abstract Background Evolution of toxicity testing is predicated upon using in vitro cell based systems to rapidly screen and predict how a chemical might cause toxicity to an organ in vivo. However, the degree to which we can extend in vitro results to in vivo activity and possible mechanisms of action remains to be fully addressed. Results Here we use the nitroaromatic 2,4,6-trinitrotoluene (TNT as a model chemical to compare and determine how we might extrapolate from in vitro data to in vivo effects. We found 341 transcripts differentially expressed in common among in vitro and in vivo assays in response to TNT. The major functional term corresponding to these transcripts was cell cycle. Similarly modulated common pathways were identified between in vitro and in vivo. Furthermore, we uncovered the conserved common transcriptional gene regulatory networks between in vitro and in vivo cellular liver systems that responded to TNT exposure, which mainly contain 2 subnetwork modules: PTTG1 and PIR centered networks. Interestingly, all 7 genes in the PTTG1 module were involved in cell cycle and downregulated by TNT both in vitro and in vivo. Conclusions The results of our investigation of TNT effects on gene expression in liver suggest that gene regulatory networks obtained from an in vitro system can predict in vivo function and mechanisms. Inhibiting PTTG1 and its targeted cell cyle related genes could be key machanism for TNT induced liver toxicity.

  8. In vitro gene regulatory networks predict in vivo function of liver

    Science.gov (United States)

    2010-01-01

    Background Evolution of toxicity testing is predicated upon using in vitro cell based systems to rapidly screen and predict how a chemical might cause toxicity to an organ in vivo. However, the degree to which we can extend in vitro results to in vivo activity and possible mechanisms of action remains to be fully addressed. Results Here we use the nitroaromatic 2,4,6-trinitrotoluene (TNT) as a model chemical to compare and determine how we might extrapolate from in vitro data to in vivo effects. We found 341 transcripts differentially expressed in common among in vitro and in vivo assays in response to TNT. The major functional term corresponding to these transcripts was cell cycle. Similarly modulated common pathways were identified between in vitro and in vivo. Furthermore, we uncovered the conserved common transcriptional gene regulatory networks between in vitro and in vivo cellular liver systems that responded to TNT exposure, which mainly contain 2 subnetwork modules: PTTG1 and PIR centered networks. Interestingly, all 7 genes in the PTTG1 module were involved in cell cycle and downregulated by TNT both in vitro and in vivo. Conclusions The results of our investigation of TNT effects on gene expression in liver suggest that gene regulatory networks obtained from an in vitro system can predict in vivo function and mechanisms. Inhibiting PTTG1 and its targeted cell cyle related genes could be key machanism for TNT induced liver toxicity. PMID:21073692

  9. In vivo EPR oximetry using an isotopically-substituted nitroxide: Potential for quantitative measurement of tissue oxygen

    Science.gov (United States)

    Weaver, John; Burks, Scott R.; Liu, Ke Jian; Kao, Joseph P. Y.; Rosen, Gerald M.

    2016-10-01

    Variations in brain oxygen (O2) concentration can have profound effects on brain physiology. Thus, the ability to quantitate local O2 concentrations noninvasively in vivo could significantly enhance understanding of several brain pathologies. However, quantitative O2 mapping in the brain has proven difficult. The electron paramagnetic resonance (EPR) spectra of nitroxides are sensitive to molecular O2 and can be used to estimate O2 concentrations in aqueous media. We recently synthesized labile-ester-containing nitroxides, such as 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 4), which accumulate in cerebral tissue after in situ hydrolysis, and thus enable spatial mapping of O2 concentrations in the mouse brain by EPR imaging. In an effort to improve O2 quantitation, we prepared 3-acetoxymethoxycarbonyl-2,2,5,5-tetra(2H3)methyl-1-(3,4,4-2H3,1-15N)pyrrolidinyloxyl (nitroxide 2), which proved to be a more sensitive probe than its normo-isotopic version for quantifying O2 in aqueous solutions of various O2 concentrations. We now demonstrate that this isotopically substituted nitroxide is ∼2-fold more sensitive in vivo than the normo-isotopic nitroxide 4. Moreover, in vitro and in vivo EPR spectral-spatial imaging results with nitroxide 2 demonstrate significant improvement in resolution, reconstruction and spectral response to local O2 concentrations in cerebral tissue. Thus, isotopic-substituted nitroxides, such as 2, are excellent sensors for in vivo O2 quantitation in tissues, such as the brain.

  10. Beyond Drosophila: RNAi in vivo and functional genomics in insects.

    Science.gov (United States)

    Bellés, Xavier

    2010-01-01

    The increasing availability of insect genomes has revealed a large number of genes with unknown functions and the resulting problem of how to discover these functions. The RNA interference (RNAi) technique, which generates loss-of-function phenotypes by depletion of a chosen transcript, can help to overcome this challenge. RNAi can unveil the functions of new genes, lead to the discovery of new functions for old genes, and find the genes for old functions. Moreover, the possibility of studying the functions of homologous genes in different species can allow comparisons of the genetic networks regulating a given function in different insect groups, thereby facilitating an evolutionary insight into developmental processes. RNAi also has drawbacks and obscure points, however, such as those related to differences in species sensitivity. Disentangling these differences is one of the main challenges in the RNAi field.

  11. Confocal Fluorescence Imaging Enables Noninvasive Quantitative Assessment of Host Cell Populations In Vivo Following Photodynamic Therapy

    Directory of Open Access Journals (Sweden)

    Soumya Mitra, Oleg Mironov, Thomas H. Foster

    2012-01-01

    Full Text Available We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH-mediated photodynamic therapy (PDT of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV administration of 1 μmol kg-1 HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1+/CD11b+ leukocytes and major histocompatibility complex class II (MHC-II+ cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1+ cells in response to therapy. The maximum accumulation of Gr1+ cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1+ cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1+ cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

  12. In Vivo Imaging of Tissue Physiological Function using EPR Spectroscopy | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    Electron paramagnetic resonance (EPR) is a technique for studying chemical species that have one or more unpaired electrons.  The current invention describes Echo-based Single Point Imaging (ESPI), a novel EPR image formation strategy that allows in vivo imaging of physiological function.  The National Cancer Institute's Radiation Biology Branch is seeking statements of capability or interest from parties interested in in-licensing an in vivo imaging using Electron paramagnetic resonance (EPR) to measure active oxygen species.

  13. The functions of laminins: lessons from in vivo studies

    DEFF Research Database (Denmark)

    Ryan, M C; Christiano, A M; Engvall, E

    1996-01-01

    diversity of the laminin family members makes highly specialized functions possible. While all laminins may share many functional properties, the individual chains are involved in interactions which cannot be substituted for by other laminins or by other basement membrane components. While this concept...... is how strongly the induced mouse mutations mimic human disease. With all the concerns with genetic background differences and species specific effects, manipulation of the laminin genes appears to be a particularly good first approach to identifying the causes of human disease. There is an abundant...

  14. Inflammation modulates human HDL composition and function in vivo

    Science.gov (United States)

    Inflammation may directly impair HDL functions, in particular reverse cholesterol transport (RCT), but limited data support this concept in humans. Our study was designed to investigate this relationship. We employed low-dose human endotoxemia to assess the effects of inflammation on HDL and RCT-rel...

  15. Using vaccinations to assess in vivo immune function in psychoneuroimmunology.

    Science.gov (United States)

    Burns, Victoria E

    2012-01-01

    Finding clinically relevant measures of immune function is an important challenge in psychoneuroimmunological research. Here, we discuss the advantages of the vaccination model, and provide guidance on the methodological decisions that are important to consider in the use of this technique. These include the choice of vaccination, timing of assessments, and the available outcome measures.

  16. Rat parotid cell function in vitro following x irradiation in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Bodner, L.; Kuyatt, B.L.; Hand, A.R.; Baum, B.J.

    1984-02-01

    The effect of X irradiation on rat parotid acinar cell function was evaluated in vitro 1, 3, and 7 days following in vivo exposure to 2000 R. Several cellular functions were followed: protein secretion (amylase release), ion movement (K/sup +/ efflux and reuptake), amino acid transport (..cap alpha..-amino(/sup 14/C)isobutyric acid), and an intermediary metabolic response ((/sup 14/C)glucose oxidation). In addition both the morphologic appearance and in vivo saliva secretory ability of parotid cells were assessed. Our results demonstrate that surviving rat parotid acinar cells, isolated and studied in vitro 1-7 days following 2000 R, remain functionally intact despite in vivo diminution of secretory function.

  17. An optimized triple modality reporter for quantitative in vivo tumor imaging and therapy evaluation.

    Science.gov (United States)

    Levin, Rachel A; Felsen, Csilla N; Yang, Jin; Lin, John Y; Whitney, Michael A; Nguyen, Quyen T; Tsien, Roger Y

    2014-01-01

    We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter. Fluorescence and bioluminescence imaging of the triple modality reporter were used to accurately quantify the therapeutic responses of MDA-MB-231 tumors to the chemotherapeutic agent monomethyl auristatin E in vivo in athymic nude mice. Positive correlation was observed between the fluorescence and bioluminescence signals, and these signals were also positively correlated with the ex vivo tumor weights. This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo.

  18. An optimized triple modality reporter for quantitative in vivo tumor imaging and therapy evaluation.

    Directory of Open Access Journals (Sweden)

    Rachel A Levin

    Full Text Available We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson, enhanced firefly luciferase enzyme (Luc2, and truncated wild type herpes simplex virus I thymidine kinase (wttk that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer were successfully transduced to express this triple modality reporter. Fluorescence and bioluminescence imaging of the triple modality reporter were used to accurately quantify the therapeutic responses of MDA-MB-231 tumors to the chemotherapeutic agent monomethyl auristatin E in vivo in athymic nude mice. Positive correlation was observed between the fluorescence and bioluminescence signals, and these signals were also positively correlated with the ex vivo tumor weights. This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo.

  19. Quantitative imaging of intracellular signaling for personalized pancreatic cancer therapy in an in vivo avatar (Conference Presentation)

    Science.gov (United States)

    Samkoe, Kimberley S.; Schultz, Emily; Park, Yeonjae; Fischer, Dawn; Pogue, Brian W.; Smith, Kerrington; Tichauer, Kenneth M.; Gibbs, Summer L.

    2017-02-01

    Pancreatic ductal adenocarcinomas (PDAC) are notoriously difficult to treat and in general, molecular targeted therapies have failed even when the targeted protein is overexpressed in the tumor tissue. Genetic mutations in extracellular receptors and downstream signaling proteins (i.e., RAS signaling pathway) and convoluted intracellular cross-talk between cell signaling pathways are likely reasons that these promising therapies fail. Monitoring the complex relationship between intracellular protein signaling is difficult and to-date, standard techniques that are used (Western blot, flow cytometry, immunohistochemistry, etc.) are invasive, static and do not accurately represent in vivo structure-function relationships. Here, we describe the development of an in ovo avatar using patient derived tumors grown on the chicken chorioallantoic membrane (CAM) and the novel fluorescence-based Quantitative Protein Expression Tracking (QUIET) methodology to bridge the gap between oncology, genomics and patient outcomes. Previously developed paired-agent imaging, was extended to a three-compartment model system in QUIET, which utilizes three types of imaging agents: novel fluorophore conjugated cell permeable targeted and untargeted small molecule paired-agents, in addition to a tumor perfusion agent that is not cell membrane permeable. We have demonstrated the ability to quantify the intracellular binding domain of a trans-membrane protein in vitro using cell permeable fluorescent agents (erlotinib-TRITC and control isotype-BODIPY FL). In addition, we have demonstrated imaging protocols to simultaneously image up to 6 spectrally distinct organic fluorophores in in ovo avatars using the Nuance EX (Perkin Elmer) and established proof-of-principle intracellular and extracellular protein concentrations of epidermal growth factor receptor using QUIET and traditional paired-agent imaging.

  20. In vivo functional neurochemistry of human cortical cholinergic function during visuospatial attention

    Science.gov (United States)

    Lindner, Michael; Bell, Tiffany; Iqbal, Somya; Mullins, Paul Gerald

    2017-01-01

    Cortical acetylcholine is involved in key cognitive processes such as visuospatial attention. Dysfunction in the cholinergic system has been described in a number of neuropsychiatric disorders. Levels of brain acetylcholine can be pharmacologically manipulated, but it is not possible to directly measure it in vivo in humans. However, key parts of its biochemical cascade in neural tissue, such as choline, can be measured using magnetic resonance spectroscopy (MRS). There is evidence that levels of choline may be an indirect but proportional measure of acetylcholine availability in brain tissue. In this study, we measured relative choline levels in the parietal cortex using functional (event-related) MRS (fMRS) during performance of a visuospatial attention task, with a modelling approach verified using simulated data. We describe a task-driven interaction effect on choline concentration, specifically driven by contralateral attention shifts. Our results suggest that choline MRS has the potential to serve as a proxy of brain acetylcholine function in humans. PMID:28192451

  1. Intravital FRET: Probing Cellular and Tissue Function in Vivo

    Directory of Open Access Journals (Sweden)

    Helena Radbruch

    2015-05-01

    Full Text Available The development of intravital Förster Resonance Energy Transfer (FRET is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice.

  2. Nucleotide-sugar transporters: structure, function and roles in vivo

    Directory of Open Access Journals (Sweden)

    Handford M.

    2006-01-01

    Full Text Available The glycosylation of glycoconjugates and the biosynthesis of polysaccharides depend on nucleotide-sugars which are the substrates for glycosyltransferases. A large proportion of these enzymes are located within the lumen of the Golgi apparatus as well as the endoplasmic reticulum, while many of the nucleotide-sugars are synthesized in the cytosol. Thus, nucleotide-sugars are translocated from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum by multiple spanning domain proteins known as nucleotide-sugar transporters (NSTs. These proteins were first identified biochemically and some of them were cloned by complementation of mutants. Genome and expressed sequence tag sequencing allowed the identification of a number of sequences that may encode for NSTs in different organisms. The functional characterization of some of these genes has shown that some of them can be highly specific in their substrate specificity while others can utilize up to three different nucleotide-sugars containing the same nucleotide. Mutations in genes encoding for NSTs can lead to changes in development in Drosophila melanogaster or Caenorhabditis elegans, as well as alterations in the infectivity of Leishmania donovani. In humans, the mutation of a GDP-fucose transporter is responsible for an impaired immune response as well as retarded growth. These results suggest that, even though there appear to be a fair number of genes encoding for NSTs, they are not functionally redundant and seem to play specific roles in glycosylation.

  3. Development and in vivo evaluation of papain-functionalized nanoparticles.

    Science.gov (United States)

    Müller, Christiane; Perera, Glen; König, Verena; Bernkop-Schnürch, Andreas

    2014-05-01

    The aim of the present study was to develop a novel nanoparticulate delivery system being capable of penetrating the intestinal mucus layer by cleaving mucoglycoprotein substructures. Nanoparticles based on papain grafted polyacrylic acid (papain-g-PAA) were prepared via ionic gelation and labeled with fluorescein diacetate. In vitro, the proteolytic potential of papain modified nanoparticles was investigated by rheological measurements and diffusion studies across fresh porcine intestinal mucus. The presence of papain on the surface and inside the particles strongly decreases viscosity of the mucus leading to facilitated particle transition across the mucus layer. Results of the permeation studies revealed that enzyme grafted particles diffuse through mucus layer to a 3.0-fold higher extent than the same particles without enzyme. Furthermore, the penetration behavior of the nanocarriers along the gastrointestinal tract of Sprague Dawley rats was investigated after oral administration of nanoparticles formulated as enteric coated capsules. The majority of the papain functionalized particles was able to traverse across the mucus layer and remained in the duodenum and jejunum of the small intestine where drug absorption primarily occurs. Polymeric nanoparticles combined with mucolytic enzymes that are capable of overcoming intestinal mucus barriers offer an encouraging new attempt for mucosal drug delivery.

  4. Staphylococcal biofilm formation on the surface of three different calcium phosphate bone grafts: a qualitative and quantitative in vivo analysis.

    Science.gov (United States)

    Furustrand Tafin, Ulrika; Betrisey, Bertrand; Bohner, Marc; Ilchmann, Thomas; Trampuz, Andrej; Clauss, Martin

    2015-03-01

    Differences in physico-chemical characteristics of bone grafts to fill bone defects have been demonstrated to influence in vitro bacterial biofilm formation. Aim of the study was to investigate in vivo staphylococcal biofilm formation on different calcium phosphate bone substitutes. A foreign-body guinea-pig infection model was used. Teflon cages prefilled with β-tricalcium phosphate, calcium-deficient hydroxyapatite, or dicalcium phosphate (DCP) scaffold were implanted subcutaneously. Scaffolds were infected with 2 × 10(3) colony-forming unit of Staphylococcus aureus (two strains) or S. epidermidis and explanted after 3, 24 or 72 h of biofilm formation. Quantitative and qualitative biofilm analysis was performed by sonication followed by viable counts, and microcalorimetry, respectively. Independently of the material, S. aureus formed increasing amounts of biofilm on the surface of all scaffolds over time as determined by both methods. For S. epidermidis, the biofilm amount decreased over time, and no biofilm was detected by microcalorimetry on the DCP scaffolds after 72 h of infection. However, when using a higher S. epidermidis inoculum, increasing amounts of biofilm were formed on all scaffolds as determined by microcalorimetry. No significant variation in staphylococcal in vivo biofilm formation was observed between the different materials tested. This study highlights the importance of in vivo studies, in addition to in vitro studies, when investigating biofilm formation of bone grafts.

  5. Functional dissection of synaptic circuits: in vivo patch-clamp recording in neuroscience

    Directory of Open Access Journals (Sweden)

    Can eTao

    2015-05-01

    Full Text Available Neuronal activity is dominated by synaptic inputs from excitatory or inhibitory neural circuits. With the development of in vivo patch-clamp recording, especially in vivo voltage-clamp recording, researchers can not only directly measure neuronal activity, such as spiking responses or membrane potential dynamics, but also quantify synaptic inputs from excitatory and inhibitory circuits in living animals. This approach enables researchers to directly unravel different synaptic components and to understand their underlying roles in particular brain functions. Combining in vivo patch-clamp recording with other techniques, such as two-photon imaging or optogenetics, can provide even clearer functional dissection of the synaptic contributions of different neurons or nuclei. Here, we summarized current applications and recent research progress using the in vivo patch-clamp recording method and focused on its role in the functional dissection of different synaptic inputs. The key factors of a successful in vivo patch-clamp experiment and possible solutions based on references and our experiences were also discussed.

  6. In vivo multiplex quantitative analysis of 3 forms of alpha melanocyte stimulating hormone in pituitary of prolyl endopeptidase deficient mice

    Directory of Open Access Journals (Sweden)

    Perroud Bertrand

    2009-06-01

    Full Text Available Abstract Background In vitro reactions are useful to identify putative enzyme substrates, but in vivo validation is required to identify actual enzyme substrates that have biological meaning. To investigate in vivo effects of prolyl endopeptidase (PREP, a serine protease, on alpha melanocyte stimulating hormone (α-MSH, we developed a new mass spectrometry based technique to quantitate, in multiplex, the various forms of α-MSH. Methods Using Multiple Reaction Monitoring (MRM, we analyzed peptide transitions to quantify three different forms of α-MSH. Transitions were first confirmed using standard peptides. Samples were then analyzed by mass spectrometry using a triple quadrupole mass spectrometer, after elution from a reverse phase C18 column by a gradient of acetonitrile. Results We first demonstrate in vitro that PREP digests biological active alpha melanocyte stimulating hormone (α-MSH1–13, by cleaving the terminal amidated valine and releasing a truncated alpha melanocyte stimulating hormone (α-MSH1–12 product – the 12 residues α-MSH form. We then use the technique in vivo to analyze the MRM transitions of the three different forms of α-MSH: the deacetylated α-MSH1–13, the acetylated α-MSH1–13 and the truncated form α-MSH1–12. For this experiment, we used a mouse model (PREP-GT in which the serine protease, prolyl endopeptidase, is deficient due to a genetrap insertion. Here we report that the ratio between acetylated α-MSH1–13 and α-MSH1–12 is significantly increased (P-value = 0.015, N = 6 in the pituitaries of PREP-GT mice when compared to wild type littermates. In addition no significant changes were revealed in the relative level of α-MSH1–13 versus the deacetylated α-MSH1–13. These results combined with the demonstration that PREP digests α-MSH1–13 in vitro, strongly suggest that α-MSH1–13 is an in vivo substrate of PREP. Conclusion The multiplex targeted quantitative peptidomics technique we

  7. Predicting in vivo glioma growth with the reaction diffusion equation constrained by quantitative magnetic resonance imaging data

    Science.gov (United States)

    Hormuth, David A., II; Weis, Jared A.; Barnes, Stephanie L.; Miga, Michael I.; Rericha, Erin C.; Quaranta, Vito; Yankeelov, Thomas E.

    2015-07-01

    Reaction-diffusion models have been widely used to model glioma growth. However, it has not been shown how accurately this model can predict future tumor status using model parameters (i.e., tumor cell diffusion and proliferation) estimated from quantitative in vivo imaging data. To this end, we used in silico studies to develop the methods needed to accurately estimate tumor specific reaction-diffusion model parameters, and then tested the accuracy with which these parameters can predict future growth. The analogous study was then performed in a murine model of glioma growth. The parameter estimation approach was tested using an in silico tumor ‘grown’ for ten days as dictated by the reaction-diffusion equation. Parameters were estimated from early time points and used to predict subsequent growth. Prediction accuracy was assessed at global (total volume and Dice value) and local (concordance correlation coefficient, CCC) levels. Guided by the in silico study, rats (n = 9) with C6 gliomas, imaged with diffusion weighted magnetic resonance imaging, were used to evaluate the model’s accuracy for predicting in vivo tumor growth. The in silico study resulted in low global (tumor volume error 0.92) and local (CCC values >0.80) level errors for predictions up to six days into the future. The in vivo study showed higher global (tumor volume error >11.7%, Dice silico study shows that model parameters can be accurately estimated and used to accurately predict future tumor growth at both the global and local scale. However, the poor predictive accuracy in the experimental study suggests the reaction-diffusion equation is an incomplete description of in vivo C6 glioma biology and may require further modeling of intra-tumor interactions including segmentation of (for example) proliferative and necrotic regions.

  8. Quantitative Analysis of HER2 Receptor Expression In Vivo by Near-Infrared Optical Imaging

    Directory of Open Access Journals (Sweden)

    Victor Chernomordik

    2010-07-01

    Full Text Available Human epidermal growth factor receptor 2 (HER2 overexpression in breast cancers is associated with poor prognosis and resistance to therapy. Current techniques for estimating this important characteristic use ex vivo assays that require tissue biopsies. We suggest a novel noninvasive method to characterize HER2 expression in vivo, using optical imaging, based on HER2-specific probes (albumin-binding domain–fused-(ZHER2:3422-Cys Affibody molecules [Affibody AB, Solna, Sweden], labeled with Alexa Fluor 750 [Molecular Probes, Invitrogen, Carlsbad, CA] that could be used concomitantly with HER2-targeted therapy. Subcutaneous tumor xenografts, expressing different levels of HER2, were imaged with a near-infrared fluorescence small-animal imaging system at several times postinjection of the probe. The compartmental ligand-receptor model was used to calculate HER2 expression from imaging data. Correlation between HER2 amplification/overexpression in tumor cells and parameters, directly estimated from the sequence of optical images, was observed (eg, experimental data for BT474 xenografts indicate that initial slope, characterizing the temporal dependence of the fluorescence intensity detected in the tumor, linearly depends on the HER2 expression, as measured ex vivo by an enzyme-linked immunosorbent assay for the same tumor. The results obtained from tumors expressing different levels of HER2 substantiate a similar relationship between the initial slope and HER2 amplification/overexpression. This work shows that optical imaging, combined with mathematical modeling, allows noninvasive monitoring of HER2 expression in vivo.

  9. Application of electrical stimulation for functional tissue engineering in vitro and in vivo

    Science.gov (United States)

    Radisic, Milica (Inventor); Park, Hyoungshin (Inventor); Langer, Robert (Inventor); Freed, Lisa (Inventor); Vunjak-Novakovic, Gordana (Inventor)

    2013-01-01

    The present invention provides new methods for the in vitro preparation of bioartificial tissue equivalents and their enhanced integration after implantation in vivo. These methods include submitting a tissue construct to a biomimetic electrical stimulation during cultivation in vitro to improve its structural and functional properties, and/or in vivo, after implantation of the construct, to enhance its integration with host tissue and increase cell survival and functionality. The inventive methods are particularly useful for the production of bioartificial equivalents and/or the repair and replacement of native tissues that contain electrically excitable cells and are subject to electrical stimulation in vivo, such as, for example, cardiac muscle tissue, striated skeletal muscle tissue, smooth muscle tissue, bone, vasculature, and nerve tissue.

  10. Novel Uses of In Vitro Data to Develop Quantitative Biological Activity Relationship Models for in Vivo Carcinogenicity Prediction.

    Science.gov (United States)

    Pradeep, Prachi; Povinelli, Richard J; Merrill, Stephen J; Bozdag, Serdar; Sem, Daniel S

    2015-04-01

    The availability of large in vitro datasets enables better insight into the mode of action of chemicals and better identification of potential mechanism(s) of toxicity. Several studies have shown that not all in vitro assays can contribute as equal predictors of in vivo carcinogenicity for development of hybrid Quantitative Structure Activity Relationship (QSAR) models. We propose two novel approaches for the use of mechanistically relevant in vitro assay data in the identification of relevant biological descriptors and development of Quantitative Biological Activity Relationship (QBAR) models for carcinogenicity prediction. We demonstrate that in vitro assay data can be used to develop QBAR models for in vivo carcinogenicity prediction via two case studies corroborated with firm scientific rationale. The case studies demonstrate the similarities between QBAR and QSAR modeling in: (i) the selection of relevant descriptors to be used in the machine learning algorithm, and (ii) the development of a computational model that maps chemical or biological descriptors to a toxic endpoint. The results of both the case studies show: (i) improved accuracy and sensitivity which is especially desirable under regulatory requirements, and (ii) overall adherence with the OECD/REACH guidelines. Such mechanism based models can be used along with QSAR models for prediction of mechanistically complex toxic endpoints. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Quantitative Correlation of in Vivo Properties with in Vitro Assay Results: The in Vitro Binding of a Biotin-DNA Analogue Modifier with Streptavidin Predicts the in Vivo Avidin-Induced Clearability of the Analogue-Modified Antibody.

    Science.gov (United States)

    Dou, Shuping; Virostko, John; Greiner, Dale L; Powers, Alvin C; Liu, Guozheng

    2015-08-03

    Quantitative prediction of in vivo behavior using an in vitro assay would dramatically accelerate pharmaceutical development. However, studies quantitatively correlating in vivo properties with in vitro assay results are rare because of the difficulty in quantitatively understanding the in vivo behavior of an agent. We now demonstrate such a correlation as a case study based on our quantitative understanding of the in vivo chemistry. In an ongoing pretargeting project, we designed a trifunctional antibody (Ab) that concomitantly carried a biotin and a DNA analogue (hereafter termed MORF). The biotin and the MORF were fused into one structure prior to conjugation to the Ab for the concomitant attachment. Because it was known that avidin-bound Ab molecules leave the circulation rapidly, this design would theoretically allow complete clearance by avidin. The clearability of the trifunctional Ab was determined by calculating the blood MORF concentration ratio of avidin-treated Ab to non-avidin-treated Ab using mice injected with these compounds. In theory, any compromised clearability should be due to the presence of impurities. In vitro, we measured the biotinylated percentage of the Ab-reacting (MORF-biotin)⊃-NH2 modifier, by addition of streptavidin to the radiolabeled (MORF-biotin)⊃-NH2 samples and subsequent high-performance liquid chromatography (HPLC) analysis. On the basis of our previous quantitative understanding, we predicted that the clearability of the Ab would be equal to the biotinylation percentage measured via HPLC. We validated this prediction within a 3% difference. In addition to the high avidin-induced clearability of the trifunctional Ab (up to ∼95%) achieved by the design, we were able to predict the required quality of the (MORF-biotin)⊃-NH2 modifier for any given in vivo clearability. This approach may greatly reduce the steps and time currently required in pharmaceutical development in the process of synthesis, chemical analysis, in

  12. In vivo quantitation of metabolite concentrations in the brain by means of proton MRS

    DEFF Research Database (Denmark)

    Henriksen, O

    1995-01-01

    MRS offers unique possibilities for non-invasive studies of biochemistry in the human brain in vivo. A growing body of evidence suggests that proton MRS may contribute to the clinical evaluation of a number of pathologies including ischaemia, tumours, epilepsy, metabolic and neuropaediatric...... (NAA), total creatine, choline containing compounds, (Cho) and inositols (Ins). Internal standards (unsaturated water signal) as well as external standards have been used for signal calibration. Quality control with respect to signal linearity with concentration or with size of selected volume......, selection efficiency, outer volume depression and signal contamination is essential for validation of the measurements. Furthermore, corrections for the influence of relaxation behavior are necessary. The results published so far indicate that the concentrations of NAA, total creatine, Cho and Ins in mmoles...

  13. In vivo MRI biocompatibility evaluation of functionalized carbon fibers in reaction with soft tissues

    Directory of Open Access Journals (Sweden)

    Prokić B.B.

    2012-01-01

    Full Text Available In modern medicine implants are very important and so is their design and choice of materials. Almost equally important is the choice of imaging technique used to in vivo monitor their fate and biocompatibility. The aim of this study was to evaluate the ability of magnetic resonance imaging (MRI in monitoring the biocompatibility of two newly designed carbon fibers. We have analyzed the interaction of surface functionalized carbon fibers (basic and acidic with muscle and subcutaneous tissues of rabbits. MRI techniques showed to be useful in longitudinal monitoring of the surrounding tissues, assessment of biocompatibility of new implants, and in the distinction of in vivo surgical edema from inflammation. Histopathology confirmed MRI results, thus showing that MRI has a great potential for in vivo studies of such materials. [Projekat Ministarstva nauke Republike Srbije, br. III 45006 and III 41005

  14. In vivo myogenic potential of human CD133+ muscle-derived stem cells: a quantitative study.

    Science.gov (United States)

    Negroni, Elisa; Riederer, Ingo; Chaouch, Soraya; Belicchi, Marzia; Razini, Paola; Di Santo, James; Torrente, Yvan; Butler-Browne, Gillian S; Mouly, Vincent

    2009-10-01

    In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle-derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2(-/-) gammaC(-/-) C5(-/-) mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle-derived CD133+ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133+/CD34+ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133+ cells could be an attractive candidate for cellular therapy.

  15. In Vivo Myogenic Potential of Human CD133+ Muscle-derived Stem Cells: A Quantitative Study

    Science.gov (United States)

    Negroni, Elisa; Riederer, Ingo; Chaouch, Soraya; Belicchi, Marzia; Razini, Paola; Di Santo, James; Torrente, Yvan; Butler-Browne, Gillian S; Mouly, Vincent

    2009-01-01

    In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle–derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2−/− γC−/− C5−/− mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle–derived CD133+ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133+/CD34+ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133+ cells could be an attractive candidate for cellular therapy. PMID:19623164

  16. In Vivo Quantitative Ultrasound Image Analysis of Femoral Subchondral Bone in Knee Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Jana Podlipská

    2013-01-01

    Full Text Available A potential of quantitative noninvasive knee ultrasonography (US for detecting changes in femoral subchondral bone related to knee osteoarthritis (OA was investigated. Thirty-nine patients referred to a knee arthroscopy underwent dynamic noninvasive US examination of the knee joint. The subchondral bone was semiautomatically segmented from representative US images of femoral medial and lateral condyles and intercondylar notch area. Subsequently, the normalized mean gray-level intensity profile, starting from the cartilage-bone interface and extending to the subchondral bone depth of ~1.7 mm, was calculated. The obtained profile was divided into 5 depth levels and the mean of each level, as well as the slope of the profile within the first two levels, was calculated. The US quantitative data were compared with the arthroscopic Noyes’ grading and radiographic Kellgren-Lawrence (K-L grading. Qualitatively, an increase in relative subchondral bone US gray-level values was observed as OA progressed. Statistically significant correlations were observed between normalized US mean intensity or intensity slope especially in subchondral bone depth level 2 and K-L grading (r=0.600, P<0.001; r=0.486, P=0.006, resp. or femoral arthroscopic scoring (r=0.332, P=0.039; r=0.335, P=0.037, resp.. This novel quantitative noninvasive US analysis technique is promising for detection of femoral subchondral bone changes in knee OA.

  17. Quantitative Phenotyping-Based In Vivo Chemical Screening in a Zebrafish Model of Leukemia Stem Cell Xenotransplantation

    Science.gov (United States)

    Zhang, Beibei; Shimada, Yasuhito; Kuroyanagi, Junya; Umemoto, Noriko; Nishimura, Yuhei; Tanaka, Toshio

    2014-01-01

    Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs) is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo) and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro), the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+) cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange) and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine). Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs. PMID:24454867

  18. A study on the quantitative evaluation of skin barrier function

    Science.gov (United States)

    Maruyama, Tomomi; Kabetani, Yasuhiro; Kido, Michiko; Yamada, Kenji; Oikaze, Hirotoshi; Takechi, Yohei; Furuta, Tomotaka; Ishii, Shoichi; Katayama, Haruna; Jeong, Hieyong; Ohno, Yuko

    2015-03-01

    We propose a quantitative evaluation method of skin barrier function using Optical Coherence Microscopy system (OCM system) with coherency of near-infrared light. There are a lot of skin problems such as itching, irritation and so on. It has been recognized skin problems are caused by impairment of skin barrier function, which prevents damage from various external stimuli and loss of water. To evaluate skin barrier function, it is a common strategy that they observe skin surface and ask patients about their skin condition. The methods are subjective judgements and they are influenced by difference of experience of persons. Furthermore, microscopy has been used to observe inner structure of the skin in detail, and in vitro measurements like microscopy requires tissue sampling. On the other hand, it is necessary to assess objectively skin barrier function by quantitative evaluation method. In addition, non-invasive and nondestructive measuring method and examination changes over time are needed. Therefore, in vivo measurements are crucial for evaluating skin barrier function. In this study, we evaluate changes of stratum corneum structure which is important for evaluating skin barrier function by comparing water-penetrated skin with normal skin using a system with coherency of near-infrared light. Proposed method can obtain in vivo 3D images of inner structure of body tissue, which is non-invasive and non-destructive measuring method. We formulate changes of skin ultrastructure after water penetration. Finally, we evaluate the limit of performance of the OCM system in this work in order to discuss how to improve the OCM system.

  19. Human in vivo regional intestinal permeability: quantitation using site-specific drug absorption data.

    Science.gov (United States)

    Sjögren, Erik; Dahlgren, David; Roos, Carl; Lennernäs, Hans

    2015-06-01

    Application of information on regional intestinal permeability has been identified as a key aspect of successful pharmaceutical product development. This study presents the results and evaluation of an approach for the indirect estimation of site-specific in vivo intestinal effective permeability (Peff) in humans. Plasma concentration-time profiles from 15 clinical studies that administered drug solutions to specific intestinal regions were collected and analyzed. The intestinal absorption rate for each drug was acquired by deconvolution, using historical intravenous data as reference, and used with the intestinal surface area and the dose remaining in the lumen to estimate the Peff. Forty-three new Peff values were estimated (15 from the proximal small intestine, 11 from the distal small intestine, and 17 from the large intestine) for 14 active pharmaceutical ingredients representing a wide range of biopharmaceutical properties. A good correlation (r(2) = 0.96, slope = 1.24, intercept = 0.030) was established between these indirect jejunal Peff estimates and jejunal Peff measurements determined directly using the single-pass perfusion double balloon technique. On average, Peff estimates from the distal small intestine and large intestine were 90% and 40%, respectively, of those from the proximal small intestine. These results support the use of the evaluated deconvolution method for indirectly estimating regional intestinal Peff in humans. This study presents the first comprehensive data set of estimated human regional intestinal permeability values for a range of drugs. These biopharmaceutical data can be used to improve the accuracy of gastrointestinal absorption predictions used in drug development decision-making.

  20. Quantitative biocompatibility evaluation of nickel-free high-nitrogen stainless steel in vitro/in vivo.

    Science.gov (United States)

    Inoue, Motoki; Sasaki, Makoto; Katada, Yasuyuki; Taguchi, Tetsushi

    2014-01-01

    Coronary stents must not provoke an inflammatory response; however, some kinds of ions that are released from biometals induce biological reaction. In the present study, we quantitatively evaluated biological reaction of nickel-free high-nitrogen stainless steel (HNS) by endothelial cell culture, and a bioimaging system using NF-κB/luciferase transgenic mice to confirm the potential of HNS for the application of coronary stent. Endothelialization was greater with HNS than with commercial stainless steel (SUS316L). In vivo inflammatory response of HNS was lower than that of SUS316L. These differences may be related to the amounts of nickel ion eluted from the stents, as HNS did not elute nickel ion. These data suggest that HNS may be useful as a material for coronary artery stents.

  1. Comparative analysis of single-species and polybacterial wound biofilms using a quantitative, in vivo, rabbit ear model.

    Directory of Open Access Journals (Sweden)

    Akhil K Seth

    Full Text Available INTRODUCTION: The recent literature suggests that chronic wound biofilms often consist of multiple bacterial species. However, without appropriate in vivo, polybacterial biofilm models, our understanding of these complex infections remains limited. We evaluate and compare the effect of single- and mixed-species biofilm infections on host wound healing dynamics using a quantitative, in vivo, rabbit ear model. METHODS: Six-mm dermal punch wounds in New Zealand rabbit ears were inoculated with Staphylococcus aureus strain UAMS-1, Pseudomonas aeruginosa strain PAO1, or both, totaling 10/6 colony-forming units/wound. Bacterial proliferation and maintenance in vivo were done using procedures from our previously published model. Wounds were harvested for histological measurement of wound healing, viable bacterial counts using selective media, or inflammatory cytokine (IL-1β, TNF-α expression via quantitative reverse-transcription PCR. Biofilm structure was studied using scanning electron microscopy (SEM. For comparison, biofilm deficient mutant UAMS-929 replaced strain UAMS-1 in some mixed-species infections. RESULTS: Bacterial counts verified the presence of both strains UAMS-1 and PAO1 in polybacterial wounds. Over time, strain PAO1 became predominant (p<0.001. SEM showed colocalization of both species within an extracellular matrix at multiple time-points. Compared to each monospecies infection, polybacterial biofilms impaired all wound healing parameters (p<0.01, and increased expression of IL-1β and TNF-α (p<0.05. In contrast, mixed-species infections using biofilm-deficient mutant UAMS-929 instead of wild-type strain UAMS-1 showed less wound impairment (p<0.01 with decreased host cytokine expression (p<0.01, despite a bacterial burden and distribution comparable to that of mixed-wild-type wounds. CONCLUSIONS: This study reveals that mixed-species biofilms have a greater impact on wound healing dynamics than their monospecies counterparts. The

  2. A Quantitative Study on the in-vitro and in-vivo Acetylation of High Mobility Group A1 Proteins

    OpenAIRE

    Zhang, Qingchun; Zhang, Kangling; Zou, Yan; Perna, Avi; Wang, Yinsheng

    2007-01-01

    High mobility group (HMG) A1 proteins are subject to a number of post-translational modifications, which may regulate their function in gene transcription and other cellular processes. We examined, by using mass spectrometry, the acetylation of HMGA1a and HMGA1b proteins induced by histone acetyltransferases p300 and PCAF in vitro and in PC-3 human prostate cancer cells in vivo. It turned out that five lysine residues in HMGA1a, i.e., Lys-14, Lys-64, Lys-66, Lys-70, and Lys-73, could be acety...

  3. In vivo quantitative evaluation of the rat retinal nerve fiber layer with optical coherence tomography.

    Science.gov (United States)

    Nagata, Atsushi; Higashide, Tomomi; Ohkubo, Shinji; Takeda, Hisashi; Sugiyama, Kazuhisa

    2009-06-01

    To determine whether optical coherence tomography (OCT) is useful for quantitative evaluation of the thickness of the rat retinal nerve fiber layer (RNFL) in an optic nerve crush model. An OCT system was developed with a modified commercial time-domain OCT and a superluminescent diode with a bandwidth of 150 nm. Optical components were optimized to acquire rat retinal images. The right optic nerve was crushed intraorbitally with a clip. The left eye served as the untreated control. Circumpapillary OCT scans with a circle diameter of 500 microm centered on the optic disc were performed before and 1, 2, and 4 weeks after the crush. Repeatability and reproducibility of RNFL thickness measurements were evaluated. The RNFL thicknesses at 400, 500, and 600 microm from the center of the optic disc determined by linear vertical OCT scans were compared with thicknesses in retinal sections. The mean RNFL thicknesses in circumpapillary OCT scans were 27.9 +/- 1.8, 29.2 +/- 2.4, 19.9 +/- 2.3, and 4.5 +/- 3.6 microm before and 1, 2, and 4 weeks after the crush, respectively. RNFL thickness was unchanged 1 week after the crush, but then decreased significantly and progressively after the second week (P < 0.01). Coefficients of repeatability and reproducibility were less than 10% except for the crushed eyes at 4 weeks. RNFL thicknesses in OCT images correlated significantly with thicknesses determined histologically (r = 0.90, P < 0.001). OCT is a useful and valuable tool for quantitative evaluation of rat RNFL thickness.

  4. SERPINE2/Protease Nexin-1 in vivo multiple functions: Does the puzzle make sense?

    Science.gov (United States)

    Monard, Denis

    2017-02-01

    Cultures of glial cells and fibroblasts allowed and lead to the identification SERPINE2/Protease Nexin-1 (SERPINE2/PN-1). Cellular, biochemical, immunological and molecular characterization substantiated its variable expression in many organs as a function of development, adult stages, pathological situations or following injury. It is not a circulating serpin, but as other members of the family, its target specificity is influenced by components of the extracellular matrix. The challenges are to identify where and when SERPINE2/PN-1 modulatory action becomes crucial or even possibly specific in a mosaic of feasible in vivo impacts. Data providing correlations are not sufficient to satisfy this aim. Genetically modified mice, or tissue derived thereof, provide interesting in vivo models to identify and study the relevance of this serpin. This review will highlight sometimes-intriguing results indicating a crucial impact of SERPINE2/PN-1, especially in the vasculature, the nervous system or the behavior of cancer cells in vivo. Data presently available will be discussed in an attempt to define general trends in the diversity of SERPINE2/PN-1 modes of action in vivo.

  5. Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics.

    Science.gov (United States)

    Yaung, Stephanie J; Deng, Luxue; Li, Ning; Braff, Jonathan L; Church, George M; Bry, Lynn; Wang, Harris H; Gerber, Georg K

    2015-03-11

    Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large-scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co-evolution of the plasmid library and E. coli genome driving increased galactose utilization. Our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.

  6. Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics.

    Science.gov (United States)

    Yaung, Stephanie J; Deng, Luxue; Li, Ning; Braff, Jonathan L; Church, George M; Bry, Lynn; Wang, Harris H; Gerber, Georg K

    2015-03-01

    Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large-scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co-evolution of the plasmid library and E. coli genome driving increased galactose utilization. Our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.

  7. In vivo functional genomic studies of sterol carrier protein-2 gene in the yellow fever mosquito.

    Science.gov (United States)

    Peng, Rong; Maklokova, Vilena I; Chandrashekhar, Jayadevi H; Lan, Que

    2011-03-18

    A simple and efficient DNA delivery method to introduce extrachromosomal DNA into mosquito embryos would significantly aid functional genomic studies. The conventional method for delivery of DNA into insects is to inject the DNA directly into the embryos. Taking advantage of the unique aspects of mosquito reproductive physiology during vitellogenesis and an in vivo transfection reagent that mediates DNA uptake in cells via endocytosis, we have developed a new method to introduce DNA into mosquito embryos vertically via microinjection of DNA vectors in vitellogenic females without directly manipulating the embryos. Our method was able to introduce inducible gene expression vectors transiently into F0 mosquitoes to perform functional studies in vivo without transgenic lines. The high efficiency of expression knockdown was reproducible with more than 70% of the F0 individuals showed sufficient gene expression suppression (mosquitoes.

  8. Emission Computed Tomography: A New Technique for the Quantitative Physiologic Study of Brain and Heart in Vivo

    Science.gov (United States)

    Phelps, M. E.; Hoffman, E. J.; Huang, S. C.; Schelbert, H. R.; Kuhl, D. E.

    1978-01-01

    Emission computed tomography can provide a quantitative in vivo measurement of regional tissue radionuclide tracer concentrations. This facility when combined with physiologic models and radioactively labeled physiologic tracers that behave in a predictable manner allow measurement of a wide variety of physiologic variables. This integrated technique has been referred to as Physiologic Tomography (PT). PT requires labeled compounds which trace physiologic processes in a known and predictable manner, and physiologic models which are appropriately formulated and validated to derive physiologic variables from ECT data. In order to effectively achieve this goal, PT requires an ECT system that is capable of performing truly quantitative or analytical measurements of tissue tracer concentrations and which has been well characterized in terms of spatial resolution, sensitivity and signal to noise ratios in the tomographic image. This paper illustrates the capabilities of emission computed tomography and provides examples of physiologic tomography for the regional measurement of cerebral and myocardial metabolic rate for glucose, regional measurement of cerebral blood volume, gated cardiac blood pools and capillary perfusion in brain and heart. Studies on patients with stroke and myocardial ischemia are also presented.

  9. Reverse gyrase functions in genome integrity maintenance by protecting DNA breaks in vivo

    DEFF Research Database (Denmark)

    Han, Wenyuan; Feng, Xu; She, Qunxin

    2017-01-01

    Reverse gyrase introduces positive supercoils to circular DNA and is implicated in genome stability maintenance in thermophiles. The extremely thermophilic crenarchaeon Sulfolobus encodes two reverse gyrase proteins, TopR1 (topoisomerase reverse gyrase 1) and TopR2, whose functions in thermophili...... genomic DNA degradation during MMS treatment, accompanied by a higher rate of cell death. Taken together, these results indicate that TopR1 probably facilitates genome integrity maintenance by protecting DNA breaks from thermo-degradation in vivo....

  10. The effects of A. pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo

    OpenAIRE

    Miller, A.N.A.; Williams, E J; Sibley, K.; Herath, S.; Lane, E.A.; Fishwick, J.; Nash, D.M.; Rycroft, A. N.; Dobson, H; Bryant, C E; Sheldon, I M

    2007-01-01

    Uterine bacterial infection after parturition causes endometritis, perturbs ovarian function and leads to infertility in cattle. Although endometritis is caused by mixed infections, endometrial pathology is associated with the presence of Arcanobacterium pyogenes. The aims of the present study were to determine the effects of A. pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo. Heat-killed A. pyogenes did not affect the production of prostaglandin F2α (PG...

  11. Preclinical In vivo Imaging for Fat Tissue Identification, Quantification, and Functional Characterization.

    Science.gov (United States)

    Marzola, Pasquina; Boschi, Federico; Moneta, Francesco; Sbarbati, Andrea; Zancanaro, Carlo

    2016-01-01

    increasing interest, will be also briefly described. For each technique the physical principles of signal detection will be overviewed and some relevant studies will be summarized. Far from being exhaustive, this review has the purpose to highlight some strategies that can be adopted for the in vivo identification, quantification, and functional characterization of adipose tissues mainly from the point of view of biophysics and physiology.

  12. Preclinical In vivo Imaging for Fat Tissue Identification, Quantification, and Functional Characterization

    Science.gov (United States)

    Marzola, Pasquina; Boschi, Federico; Moneta, Francesco; Sbarbati, Andrea; Zancanaro, Carlo

    2016-01-01

    increasing interest, will be also briefly described. For each technique the physical principles of signal detection will be overviewed and some relevant studies will be summarized. Far from being exhaustive, this review has the purpose to highlight some strategies that can be adopted for the in vivo identification, quantification, and functional characterization of adipose tissues mainly from the point of view of biophysics and physiology.

  13. Quantitative phenotyping-based in vivo chemical screening in a zebrafish model of leukemia stem cell xenotransplantation.

    Directory of Open Access Journals (Sweden)

    Beibei Zhang

    Full Text Available Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro, the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+ cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine. Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs.

  14. In vivo functional genomic studies of sterol carrier protein-2 gene in the yellow fever mosquito.

    Directory of Open Access Journals (Sweden)

    Rong Peng

    Full Text Available A simple and efficient DNA delivery method to introduce extrachromosomal DNA into mosquito embryos would significantly aid functional genomic studies. The conventional method for delivery of DNA into insects is to inject the DNA directly into the embryos. Taking advantage of the unique aspects of mosquito reproductive physiology during vitellogenesis and an in vivo transfection reagent that mediates DNA uptake in cells via endocytosis, we have developed a new method to introduce DNA into mosquito embryos vertically via microinjection of DNA vectors in vitellogenic females without directly manipulating the embryos. Our method was able to introduce inducible gene expression vectors transiently into F0 mosquitoes to perform functional studies in vivo without transgenic lines. The high efficiency of expression knockdown was reproducible with more than 70% of the F0 individuals showed sufficient gene expression suppression (<30% of the controls' levels. At the cohort level, AeSCP-2 expression knockdown in early instar larvae resulted in detectable phenotypes of the expression deficiency such as high mortality, lowered fertility, and distorted sex ratio after induction of AeSCP-2 siRNA expression in vivo. The results further confirmed the important role of AeSCP-2 in the development and reproduction of A. aegypti. In this study, we proved that extrachromosomal transient expression of an inducible gene from a DNA vector vertically delivered via vitellogenic females can be used to manipulate gene expression in F0 generation. This new method will be a simple and efficient tool for in vivo functional genomic studies in mosquitoes.

  15. Renal Cortical Lactate Dehydrogenase: A Useful, Accurate, Quantitative Marker of In Vivo Tubular Injury and Acute Renal Failure.

    Directory of Open Access Journals (Sweden)

    Richard A Zager

    Full Text Available Studies of experimental acute kidney injury (AKI are critically dependent on having precise methods for assessing the extent of tubular cell death. However, the most widely used techniques either provide indirect assessments (e.g., BUN, creatinine, suffer from the need for semi-quantitative grading (renal histology, or reflect the status of residual viable, not the number of lost, renal tubular cells (e.g., NGAL content. Lactate dehydrogenase (LDH release is a highly reliable test for assessing degrees of in vitro cell death. However, its utility as an in vivo AKI marker has not been defined. Towards this end, CD-1 mice were subjected to graded renal ischemia (0, 15, 22, 30, 40, or 60 min or to nephrotoxic (glycerol; maleate AKI. Sham operated mice, or mice with AKI in the absence of acute tubular necrosis (ureteral obstruction; endotoxemia, served as negative controls. Renal cortical LDH or NGAL levels were assayed 2 or 24 hrs later. Ischemic, glycerol, and maleate-induced AKI were each associated with striking, steep, inverse correlations (r, -0.89 between renal injury severity and renal LDH content. With severe AKI, >65% LDH declines were observed. Corresponding prompt plasma and urinary LDH increases were observed. These observations, coupled with the maintenance of normal cortical LDH mRNA levels, indicated the renal LDH efflux, not decreased LDH synthesis, caused the falling cortical LDH levels. Renal LDH content was well maintained with sham surgery, ureteral obstruction or endotoxemic AKI. In contrast to LDH, renal cortical NGAL levels did not correlate with AKI severity. In sum, the above results indicate that renal cortical LDH assay is a highly accurate quantitative technique for gauging the extent of experimental acute ischemic and toxic renal injury. That it avoids the limitations of more traditional AKI markers implies great potential utility in experimental studies that require precise quantitation of tubule cell death.

  16. Toward a quantitative analysis of in vivo proton magnetic resonance spectroscopic signals using the continuous Morlet wavelet transform

    Science.gov (United States)

    Suvichakorn, A.; Ratiney, H.; Bucur, A.; Cavassila, S.; Antoine, J. P.

    2009-10-01

    We apply the Morlet wavelet transform (MWT) for quantitatively analyzing proton magnetic resonance spectroscopic (MRS) signals, more precisely signals acquired at short echo time. These signals contain many resonating components whose frequencies are characteristic of the observed metabolites, and amplitudes are directly related to the concentrations of these metabolites. With these powerful properties, in vivo MRS can be considered as a unique non-invasive tool to explore biochemical compounds of living tissues. However, the analysis and quantification of these metabolite contributions are difficult due to the low signal-to-noise ratio, the number of overlapping frequencies and the contamination of the signal of interest with water and a baseline originating from macromolecules and lipids. The baseline is a major obstacle for MRS quantification as its shape and intensity are generally not known a priori. In this paper, we present the methodology to quantify the signals by the MWT. We assess the ability of the proposed method to recover parameters such as metabolite amplitudes, frequencies and damping factors while facing successively quantification challenges arising from the non-Lorentzian lineshapes, overlapping frequencies, and noise or baseline. Tests of the method are performed on simulated signals alone or combined with either in vitro acquisition and/or in vivo macromolecular signal acquired on a horizontal 4.7 T scanner. In presence of the macromolecules, the amplitude parameter is correctly derived by the method, thanks to the time-scale representation of the wavelet which enables us to distinguish the two signals by their time decays and without any additional pre-processing.

  17. Ospemifene metabolism in humans in vitro and in vivo: metabolite identification, quantitation, and CYP assignment of major hydroxylations.

    Science.gov (United States)

    Tolonen, Ari; Koskimies, Pasi; Turpeinen, Miia; Uusitalo, Jouko; Lammintausta, Risto; Pelkonen, Olavi

    2013-01-01

    The metabolism of ospemifene, a novel nonsteroidal selective estrogen receptor modulator, was investigated as part of its development. Metabolite identification, tentative quantitation, and CYP assignment of ospemifene were performed in human liver microsomes or homogenate incubations and in plasma samples from volunteer humans. The potential contributions of CYP enzymes were determined by recombinant human CYPs. Metabolite identification and tentative quantification were performed by liquid chromatography-mass spectrometry. The relative abundances of metabolites produced were dependent on ospemifene concentration and liver preparation, but the largest quantities of 4- and 4'-hydroxy-ospemifene (and their glucuronides in smaller quantities) were produced in human liver microsomes at low ospemifene concentrations. Other metabolites were detected in in vitro incubation with human liver including a direct glucuronide of ospemifene and some metabolites with only minor abundance. In human plasma samples, 4-hydroxy-ospemifene was the most abundant metabolite, representing about 25% of the abundance of the parent compound. All the other metabolites detected in plasma, including 4'-hydroxy-ospemifene, represented <7% of the abundance of ospemifene. Several CYP enzymes participated in 4-hydroxylation, including CYP2C9, CYP2C19, CYP2B6, and CYP3A4, whereas CYP3A enzymes were the only ones to catalyze 4'-hydroxylation. In vitro incubations with liver preparations provided a rather reliable starting point in the search for potential metabolites in clinical settings. The in vitro metabolite profile is informative for the in vivo metabolite profile, especially regarding the major hydroxylated metabolites. However, it is anticipated that extended in vivo exposures may result in an increased production of more distal metabolites from major metabolites.

  18. Quantitative amino acid profiling and stable isotopically labeled amino acid tracer enrichment used for in vivo human systemic and tissue kinetics measurements.

    Science.gov (United States)

    Bornø, Andreas; van Hall, Gerrit

    2014-03-01

    An important area within clinical functional metabolomics is in vivo amino acid metabolism and protein turnover measurements for which accurate amino acid concentrations and stable isotopically labeled amino acid enrichments are mandatory not the least when tissue metabolomics is determined. The present study describes a new sensitive liquid chromatography tandem mass-spectrometry method quantifying 20 amino acids and their tracer(s) ([ring-(13)C6]/D5Phenylalanine) in human plasma and skeletal muscle specimens. Before analysis amino acids were extracted and purified via deprotonization/ion exchange, derivatized using a phenylisothiocyanate reagent and each amino acid was quantitated with its own stable isotopically labeled internal standard (uniformly labeled-(13)C/(15)N). The method was validated according to general recommendations for chromatographic analytical methods. The calibration curve correlations for amino acids were on average; r(2)=0.998. Interday accuracy for amino acids determined in spiked plasma was on average 97.3% and the coefficient of variation (CV) was 2.6%. The ([ring-(13)C6]/D5Phenylalanine) enrichment CV's for machine reproducibility in muscle tissue fluid and plasma were 4.4 and 0.8%, and the interday variability was 3.4% and the recovery was 90.5%, respectively. In conclusion, we have developed and validated a method for quantitative amino acid profiling that meets the requirements for systemic and tissue human in vivo amino acid and protein turnover kinetics measurements. Moreover, citrulline, ornithine, π-methyl-histidine, τ-methyl-l-histidine, hydroxy-proline and carnitine were analysed but when similar precision and accuray are required an additional stable istopically labeled internal standard for these meatablites should be be added.

  19. Longitudinal in vivo muscle function analysis of the DMSXL mouse model of myotonic dystrophy type 1.

    Science.gov (United States)

    Decostre, Valérie; Vignaud, Alban; Matot, Béatrice; Huguet, Aline; Ledoux, Isabelle; Bertil, Emilie; Gjata, Bernard; Carlier, Pierre G; Gourdon, Geneviève; Hogrel, Jean-Yves

    2013-12-01

    Myotonic dystrophy is the most common adult muscle dystrophy. In view of emerging therapies, which use animal models as a proof of principle, the development of reliable outcome measures for in vivo longitudinal study of mouse skeletal muscle function is becoming crucial. To satisfy this need, we have developed a device to measure ankle dorsi- and plantarflexion torque in rodents. We present an in vivo 8-month longitudinal study of the contractile properties of the skeletal muscles of the DMSXL mouse model of myotonic dystrophy type 1. Between 4 and 12 months of age, we observed a reduction in muscle strength in the ankle dorsi- and plantarflexors of DMSXL compared to control mice although the strength per muscle cross-section was normal. Mild steady myotonia but no abnormal muscle fatigue was also observed in the DMSXL mice. Magnetic resonance imaging and histological analysis performed at the end of the study showed respectively reduced muscle cross-section area and smaller muscle fibre diameter in DMSXL mice. In conclusion, our study demonstrates the feasibility of carrying out longitudinal in vivo studies of muscle function over several months in a mouse model of myotonic dystrophy confirming the feasibility of this method to test preclinical therapeutics.

  20. Homeostasis and function of regulatory T cells (Tregs) in vivo: lessons from TCR-transgenic Tregs

    Science.gov (United States)

    Attridge, Kesley; Walker, Lucy S K

    2014-01-01

    The identification of CD25 and subsequently Forkhead box protein 3 (Foxp3) as markers for regulatory T cells (Tregs) has revolutionized our ability to explore this population experimentally. In a similar vein, our understanding of antigen-specific Treg responses in vivo owes much to the fortuitous generation of T-cell receptor (TCR)-transgenic Tregs. This has permitted tracking of Tregs with a defined specificity in vivo, facilitating analysis of how encounter with cognate antigen shapes Treg homeostasis and function. Here, we review the key lessons learned from a decade of analysis of TCR-transgenic Tregs and set this in the broader context of general progress in the field. Use of TCR-transgenic Tregs has led to an appreciation that Tregs are a highly dynamic proliferative population in vivo, rather than an anergic population as they were initially portrayed. It is now clear that Treg homeostasis is positively regulated by encounter with self-antigen expressed on peripheral tissues, which is likely to be relevant to the phenomenon of peripheral repertoire reshaping that has been described for Tregs and the observation that the Treg TCR specificities vary by anatomical location. Substantial evidence has also accumulated to support the role of CD28 costimulation and interleukin-2 in Treg homeostasis. The availability of TCR-transgenic Tregs has enabled analysis of Treg populations that are sufficient or deficient in particular genes, without the comparison being confounded by repertoire alterations. This approach has yielded insights into genes required for Treg function in vivo, with particular progress being made on the role of ctla-4 in this context. As the prospect of manipulating Treg populations in the clinic becomes reality, a full appreciation of the rules governing their homeostasis will prove increasingly important. PMID:24712457

  1. In Vivo Models to Address the Function of Polycomb Group Proteins.

    Science.gov (United States)

    Bantignies, Frédéric

    2016-01-01

    Initially discovered as repressors of homeotic gene expression in Drosophila, Polycomb group (PcG) proteins have now been shown to be involved in a plethora of biological processes. Indeed, by repressing a large number of target genes, including specific lineage genes, these chromatin factors play major roles in a multitude of cellular functions, such as pluripotency, differentiation, reprogramming, tissue regeneration, and nuclear organization. In this book chapter are presented in vivo approaches and technologies, which have been used in both mammalian and Drosophila systems to study the cellular functions of Polycomb group proteins.

  2. Quantitative in vivo CT arthrography of the human osteoarthritic knee to estimate cartilage sulphated glycosaminoglycan content: correlation with ex-vivo reference standards

    NARCIS (Netherlands)

    Tiel, J. van; Siebelt, M.; Reijman, M.; Bos, P.K.; Waarsing, J.H.; Zuurmond, A.M.; Nasserinejad, K.; Osch, G.J.V.M. van; Verhaar, J.A.N.; Krestin, G.P.; Weinans, H.; Oei, E.H.G.

    2016-01-01

    Objective. Recently, computed tomography arthrography (CTa) was introduced as quantitative imaging biomarker to estimate cartilage sulphated glycosaminoglycan (sGAG) content in human cadaveric knees. Our aim was to assess the correlation between in vivo CTa in human osteoarthritis (OA) knees and ex

  3. In-vivo imaging of the photoreceptor mosaic in retinal dystrophies and correlations with visual function

    Energy Technology Data Exchange (ETDEWEB)

    Choi, S; Doble, N; Hardy, J; Jones, S; Keltner, J; Olivier, S; Werner, J S

    2005-10-26

    To relate in-vivo microscopic retinal changes to visual function assessed with clinical tests in patients with various forms of retinal dystrophies. The UC Davis Adaptive Optics (AO) Fundus Camera was used to acquire in-vivo retinal images at the cellular level. Visual function tests, consisting of visual field analysis, multifocal electroretinography (mfERG), contrast sensitivity and color vision measures, were performed on all subjects. Five patients with different forms of retinal dystrophies and three control subjects were recruited. Cone densities were quantified for all retinal images. In all images of diseased retinas, there were extensive areas of dark space between groups of photoreceptors, where no cone photoreceptors were evident. These irregular features were not seen in healthy retinas, but were characteristic features in fundi with retinal dystrophies. There was a correlation between functional vision loss and the extent to which the irregularities occurred in retinal images. Cone densities were found to decrease with an associated decrease in retinal function. AO fundus photography is a reliable technique for assessing and quantifying the changes in the photoreceptor layer as disease progresses. Furthermore, this technique can be useful in cases where visual function tests give borderline or ambiguous results, as it allows visualization of individual photoreceptors.

  4. In vivo Pharmacological Evaluations of Pilocarpine-Loaded Antioxidant-Functionalized Biodegradable Thermogels in Glaucomatous Rabbits

    Science.gov (United States)

    Chou, Shih-Feng; Luo, Li-Jyuan; Lai, Jui-Yang

    2017-02-01

    To alleviate oxidative stress-induced ocular hypertension, grafting of antioxidant molecules to drug carriers enables a dual-function mechanism to effectively treat glaucomatous intraocular pressure (IOP) dysregulation. Providing potential application for intracameral administration of antiglaucoma medications, this study, for the first time, aims to examine in vivo pharmacological efficacy of pilocarpine-loaded antioxidant-functionalized biodegradable thermogels in glaucomatous rabbits. A series of gallic acid (GA)-grafted gelatin-g-poly(N-isopropylacrylamide) (GN) polymers were synthesized via redox reactions at 20–50 °C. Our results showed that raising redox radical initiation reaction temperature maximizes GA grafting level, antioxidant activity, and water content at 40 °C. Meanwhile, increase in overall hydrophilicity of GNGA carriers leads to fast polymer degradation and early pilocarpine depletion in vivo, which is disadvantageous to offer necessary pharmacological performance at prolonged time. By contrast, sustained therapeutic drug concentrations in aqueous humor can be achieved for long-term (i.e., 28 days) protection against corneal aberration and retinal injury after pilocarpine delivery using dual-function optimized carriers synthesized at 30 °C. The GA-functionalized injectable hydrogels are also found to contribute significantly to enhancement of retinal antioxidant defense system and preservation of histological structure and electrophysiological function, thereby supporting the benefits of drug-containing antioxidant biodegradable thermogels to prevent glaucoma development.

  5. Stiffened yeast telomerase RNA supports RNP function in vitro and in vivo.

    Science.gov (United States)

    Lebo, Kevin J; Zappulla, David C

    2012-09-01

    The 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, in addition to providing a 16-nt template region for reverse transcription, has been proposed to act as a scaffold for protein subunits. Although accessory subunits of the telomerase ribonucleoprotein (RNP) complex function even when their binding sites are relocated on the yeast telomerase RNA, the physical nature of the RNA scaffold has not been directly analyzed. Here we explore the structure-function organization of the yeast telomerase RNP by extensively stiffening the three long arms of TLC1, which connect essential and important accessory protein subunits Ku, Est1, and Sm(7), to its central catalytic hub. This 956-nt triple-stiff-arm TLC1 (TSA-T) reconstitutes active telomerase with TERT (Est2) in vitro. Furthermore, TSA-T functions in vivo, even maintaining longer telomeres than TLC1 on a per RNA basis. We also tested functional contributions of each stiffened arm within TSA-T and found that the stiffened Est1 and Ku arms contribute to telomere lengthening, while stiffening the terminal arm reduces telomere length and telomerase RNA abundance. The fact that yeast telomerase tolerates significant stiffening of its RNA subunit in vivo advances our understanding of the architectural and functional organization of this RNP and, more broadly, our conception of the world of lncRNPs.

  6. In vivo functional analysis of the human NF2 tumor suppressor gene in Drosophila.

    Directory of Open Access Journals (Sweden)

    Heather S Gavilan

    Full Text Available The proper control of tissue growth is essential during normal development and an important problem in human disease. Merlin, the product of the Neurofibromatosis 2 tumor suppressor gene, has been extensively studied to understand its functions in growth control. Here we describe experiments in which we used Drosophila as an in vivo system to test the functions of the normal human NF2 gene products and patient-derived mutant alleles. Although the predominant NF2 gene isoform, isoform 1, could functionally replace the Drosophila Merlin gene, a second isoform with a distinct C-terminal tail could not. Immunofluorescence studies show that the two isoforms have distinct subcellular localizations when expressed in the polarized imaginal epithelium, and function in genetic rescue assays correlates with apical localization of the NF2 protein. Interestingly, we found that a patient-derived missense allele, NF2L64P, appears to be temperature sensitive. These studies highlight the utility of Drosophila for in vivo functional analysis of highly conserved human disease genes.

  7. In-vivo quantification of natural incipient caries lesions using the quantitative light-induced fluoroscence method: a reproducibility study

    Science.gov (United States)

    Tranaeus, Sofia; Shi, Xie-Qi; Trollsas, Karin; Lindgren, Lars-Erik; Angmar-Mansson, Birgit

    2000-03-01

    A new method for detection and quantification of natural incipient caries lesions, the Quantitative Light-induced Fluorescence method (QLF), has recently been developed. The aim of this study was to test the repeatability and reproducibility of the analytical part of the method. In vivo captured images (CCD-video camera, Panasonic WV-KS 152, with an argon ion laser as light source) of 15 different incipient caries lesions on smooth surfaces were analyzed by three analysts. The images were analyzed three times in a randomized order, twice for the first reconstructed area (P1A1 and P1A2), and then once for a second one (P2A1). Three parameters were measured, lesion area (mm2), average change in fluorescence (%), and maximum change in fluorescence (%) in the lesion. Repeated measures ANOVA were used to calculate the intra-, and inter-examiner reliability. Intra-examiner reliability for all three analysts showed an intra-class correlation coefficient, R, between 0.93 and 0.99 (for the analyses with the first patch, P1A1 and P1A2, as well as between the first and the second patch, P1A1 and P2A1). Inter-examiner reliability showed an inter-class correlation coefficient, R, between 0.95 and 0.99 (for analyses P1A1, P1A2 and P2A1). It was concluded that the Quantitative Light- induced fluorescence method showed excellent repeatability and reproducibility concerning the analytical part of the method.

  8. In vivo quantitative evaluation of vascular parameters for angiogenesis based on sparse principal component analysis and aggregated boosted trees

    Science.gov (United States)

    Zhao, Fengjun; Liu, Junting; Qu, Xiaochao; Xu, Xianhui; Chen, Xueli; Yang, Xiang; Cao, Feng; Liang, Jimin; Tian, Jie

    2014-12-01

    To solve the multicollinearity issue and unequal contribution of vascular parameters for the quantification of angiogenesis, we developed a quantification evaluation method of vascular parameters for angiogenesis based on in vivo micro-CT imaging of hindlimb ischemic model mice. Taking vascular volume as the ground truth parameter, nine vascular parameters were first assembled into sparse principal components (PCs) to reduce the multicolinearity issue. Aggregated boosted trees (ABTs) were then employed to analyze the importance of vascular parameters for the quantification of angiogenesis via the loadings of sparse PCs. The results demonstrated that vascular volume was mainly characterized by vascular area, vascular junction, connectivity density, segment number and vascular length, which indicated they were the key vascular parameters for the quantification of angiogenesis. The proposed quantitative evaluation method was compared with both the ABTs directly using the nine vascular parameters and Pearson correlation, which were consistent. In contrast to the ABTs directly using the vascular parameters, the proposed method can select all the key vascular parameters simultaneously, because all the key vascular parameters were assembled into the sparse PCs with the highest relative importance.

  9. Quantitation of normal metabolite concentrations in six brain regions by in-vivo 1 H-MR spectroscopy

    Directory of Open Access Journals (Sweden)

    Minati Ludovico

    2010-01-01

    Full Text Available This study examined the concentrations of brain metabolites visible to in-vivo 1 H-Magnetic Resonance Spectroscopy ( 1 H-MRS at 1.5 T in a sample of 28 normal subjects. Quantitation was attempted for inositol compounds, choline units, total creatine and N-acetyl moieties, using open-source software. Six brain regions were considered: frontal and parietal white matter, medial temporal lobe, thalamus, pons and cerebellum. Absolute concentrations were derived using tissue water as an internal reference and using an external reference; metabolite signal intensity ratios with respect to creatine were also calculated. The inter-individual variability was smaller for absolute concentrations (internal reference as compared to that for signal intensity ratios. Significant regional variability in concentration was found for all metabolites, indicating that separate normative values are needed for different brain regions. The values obtained in this study can be used as reference in future studies, provided the same methodology is followed; it is confirmed that despite unsuccessful attempts in the past, smaller coefficients of variation can indeed be obtained through absolute quantification.

  10. CRISPR/Cas9 Promotes Functional Study of Testis Specific X-Linked Gene In Vivo.

    Directory of Open Access Journals (Sweden)

    Minyan Li

    Full Text Available Mammalian spermatogenesis is a highly regulated multistage process of sperm generation. It is hard to uncover the real function of a testis specific gene in vitro since the in vitro model is not yet mature. With the development of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 system, we can now rapidly generate knockout mouse models of testis specific genes to study the process of spermatogenesis in vivo. SYCP3-like X-linked 2 (SLX2 is a germ cell specific component, which contains a Cor1 domain and belongs to the XLR (X-linked, lymphocyte regulated family. Previous studies suggested that SLX2 might play an important role in mouse spermatogenesis based on its subcellular localization and interacting proteins. However, the function of SLX2 in vivo is still elusive. Here, to investigate the functions of SLX2 in spermatogenesis, we disrupted the Slx2 gene by using the CRISPR/Cas9 system. Since Slx2 is a testis specific X-linked gene, we obtained knockout male mice in the first generation and accelerated the study process. Compared with wild-type mice, Slx2 knockout mice have normal testis and epididymis. Histological observation of testes sections showed that Slx2 knockout affected none of the three main stages of spermatogenesis: mitosis, meiosis and spermiogenesis. In addition, we further confirmed that disruption of Slx2 did not affect the number of spermatogonial stem cells, meiosis progression or XY body formation by immunofluorescence analysis. As spermatogenesis was normal in Slx2 knockout mice, these mice were fertile. Taken together, we showed that Slx2 itself is not an essential gene for mouse spermatogenesis and CRISPR/Cas9 technique could speed up the functional study of testis specific X-linked gene in vivo.

  11. Techniques and evaluation from a cross-platform imaging comparison of quantitative ultrasound parameters in an in vivo rodent fibroadenoma model.

    Science.gov (United States)

    Wirtzfeld, Lauren A; Nam, Kibo; Labyed, Yassin; Ghoshal, Goutam; Haak, Alexander; Sen-Gupta, Ellora; He, Zhi; Hirtz, Nathaniel R; Miller, Rita J; Sarwate, Sandhya; Simpson, Douglas G; Zagzebski, James A; Bigelow, Timothy A; Oelze, Michael; Hall, Timothy J; O'Brien, William D

    2013-07-01

    This contribution demonstrates that quantitative ultrasound (QUS) capabilities are platform independent, using an in vivo model. Frequency-dependent attenuation estimates, backscatter coefficient, and effective scatterer diameter estimates are shown to be comparable across four different ultrasound imaging systems with varied processing techniques. The backscatter coefficient (BSC) is a fundamental material property from which several QUS parameters are estimated; therefore, consistent BSC estimates among different systems must be demonstrated. This study is an intercomparison of BSC estimates acquired by three research groups (UIUC, UW, ISU) from four in vivo spontaneous rat mammary fibroadenomas using three clinical array systems and a single-element laboratory scanner system. Because of their highly variable backscatter properties, fibroadenomas provided an extreme test case for BSC analysis, and the comparison is across systems for each tumor, not across the highly heterogeneous tumors. RF echo data spanning the 1 to 12 MHz frequency range were acquired in three dimensions from all animals using each system. Each research group processed their RF data independently, and the resulting attenuation, BSC, and effective scatterer diameter (ESD) estimates were compared. The attenuation estimates across all systems showed the same trends and consistently fit the power-law dependence on frequency. BSCs varied among the multiple slices of data acquired by each transducer, with variations between transducers being of a similar magnitude as those from slice to slice. Variation between BSC estimates was assessed via functional signal-to-noise ratios derived from backscatter data. These functional signal-to-noise ratios indicated that BSC versus frequency variations between systems ranged from negligible compared with the noise level to roughly twice the noise level. The corresponding functional analysis of variance (fANOVA) indicated statistically significant differences

  12. Analysis of in vitro and in vivo function of total knee replacements using dynamic contact models

    Science.gov (United States)

    Zhao, Dong

    Despite the high incidence of osteoarthritis in human knee joint, its causes remain unknown. Total knee replacement (TKR) has been shown clinically to be effective in restoring the knee function. However, wear of ultra-high molecular weight polyethylene has limited the longevity of TKRs. To address these important issues, it is necessary to investigate the in vitro and in vivo function of total knee replacements using dynamic contact models. A multibody dynamic model of an AMTI knee simulator was developed. Incorporating a wear prediction model into the contact model based on elastic foundation theory enables the contact surface to take into account creep and wear during the dynamic simulation. Comparisons of the predicted damage depth, area, and volume lost with worn retrievals from a physical machine were made to validate the model. In vivo tibial force distributions during dynamic and high flexion activities were investigated using the dynamic contact model. In vivo medial and lateral contact forces experienced by a well-aligned instrumented knee implant, as well as upper and lower bounds on contact pressures for a variety of activities were studied. For all activities, the predicted medial and lateral contact forces were insensitive to the selected material model. For this patient, the load split during the mid-stance phase of gait and during stair is more equal than anticipated. The external knee adduction torque has been proposed as a surrogate measure for medial compartment load during gait. However, a direct link between these two quantities has not been demonstrated using in vivo measurement of medial compartment load. In vivo data collected from a subject with an instrumented knee implant were analyzed to evaluate this link. The subject performed five different overground gait motions (normal, fast, slow, wide, and toe out) while instrumented implant, video motion, and ground reaction data were simultaneously collected. The high correlation coefficient

  13. Cholesterol lowering modulates T cell function in vivo and in vitro.

    Directory of Open Access Journals (Sweden)

    Kuang-Yuh Chyu

    Full Text Available The lipid milleu exacerbates the inflammatory response in atherosclerosis but its effect on T cell mediated immune response has not been fully elucidated. We hypothesized that lipid lowering would modulate T cell mediated immune function.T cells isolated from human PBMC or splenic T cells from apoE-/- mouse had higher proliferative response to T cell receptor (TCR ligation in medium supplemented with 10% fetal bovine serum (FBS compared to medium with 10% delipidated FBS. The differences in proliferation were associated with changes in lipid rafts, cellular cholesterol content, IL-10 secretion and subsequent activation of signaling molecule activated by TCR ligation. Immune biomarkers were also assessed in vivo using male apoE-/- mice fed atherogenic diet (AD starting at 7 weeks of age. At 25 weeks of age, a sub-group was switched to normal diet (ND whereas the rest remained on AD until euthanasia at 29 weeks of age. Dietary change resulted in a lower circulating level of cholesterol, reduced plaque size and inflammatory phenotype of plaques. These changes were associated with reduced intracellular IL-10 and IL-12 expression in CD4+ and CD8+ T cells.Our results show that lipid lowering reduces T cell proliferation and function, supporting the notion that lipid lowering modulates T cell function in vivo and in vitro.

  14. TYK2 kinase activity is required for functional type I interferon responses in vivo.

    Directory of Open Access Journals (Sweden)

    Michaela Prchal-Murphy

    Full Text Available Tyrosine kinase 2 (TYK2 is a member of the Janus kinase (JAK family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2(K923E mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein's stability. An inhibitory function was only observed upon over-expression of TYK2(K923Ein vitro. Tyk2(K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors.

  15. Exposure-in-vivo containing interventions to improve work functioning of workers with anxiety disorder : a systematic review

    NARCIS (Netherlands)

    Noordik, Erik; van der Klink, Jac J. L.; Klingen, Elmer F.; Nieuwenhuijsen, Karen; van Dijk, Frank J. H.

    2010-01-01

    Background: Anxiety disorders are associated with functional disability, sickness absence, and decreased productivity. Effective treatments of anxiety disorders can result in remission of symptoms. However the effects on work related outcomes are largely unknown. Exposure in vivo is potentially well

  16. Exposure-in-vivo containing interventions to improve work functioning of workers with anxiety disorder : a systematic review

    NARCIS (Netherlands)

    Noordik, Erik; van der Klink, Jac J. L.; Klingen, Elmer F.; Nieuwenhuijsen, Karen; van Dijk, Frank J. H.

    2010-01-01

    Background: Anxiety disorders are associated with functional disability, sickness absence, and decreased productivity. Effective treatments of anxiety disorders can result in remission of symptoms. However the effects on work related outcomes are largely unknown. Exposure in vivo is potentially well

  17. In vivo protein trapping produces a functional expression codex of the vertebrate proteome.

    Science.gov (United States)

    Clark, Karl J; Balciunas, Darius; Pogoda, Hans-Martin; Ding, Yonghe; Westcot, Stephanie E; Bedell, Victoria M; Greenwood, Tammy M; Urban, Mark D; Skuster, Kimberly J; Petzold, Andrew M; Ni, Jun; Nielsen, Aubrey L; Patowary, Ashok; Scaria, Vinod; Sivasubbu, Sridhar; Xu, Xiaolei; Hammerschmidt, Matthias; Ekker, Stephen C

    2011-06-01

    We describe a conditional in vivo protein-trap mutagenesis system that reveals spatiotemporal protein expression dynamics and can be used to assess gene function in the vertebrate Danio rerio. Integration of pGBT-RP2.1 (RP2), a gene-breaking transposon containing a protein trap, efficiently disrupts gene expression with >97% knockdown of normal transcript amounts and simultaneously reports protein expression for each locus. The mutant alleles are revertible in somatic tissues via Cre recombinase or splice-site-blocking morpholinos and are thus to our knowledge the first systematic conditional mutant alleles outside the mouse model. We report a collection of 350 zebrafish lines that include diverse molecular loci. RP2 integrations reveal the complexity of genomic architecture and gene function in a living organism and can provide information on protein subcellular localization. The RP2 mutagenesis system is a step toward a unified 'codex' of protein expression and direct functional annotation of the vertebrate genome.

  18. Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspension- cultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene TM1, TM2, TM3 and AM1 increased uidA GUS gene expression level approximately 1.5-fold, 5-fold, 1.35-fold, 1.3-fold respectively and AM2 has no effect on gene expression. TM2 was found to be a strong MAR that could effectively increase gene expression level and could be used as an effective enhancing element to construct high efficient expression vectors. In this note the relations among the sequence features, binding strength in vitro and function in vivo of the five MARs were analyzed, and the potential significance of TM2 in plant genetic engineering was dis- cussed.

  19. Allele compensation in tip60+/- mice rescues white adipose tissue function in vivo.

    Science.gov (United States)

    Gao, Yuan; Hamers, Nicole; Rakhshandehroo, Maryam; Berger, Ruud; Lough, John; Kalkhoven, Eric

    2014-01-01

    Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis), a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/- mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/- mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/- displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/- mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice.

  20. Allele compensation in tip60+/- mice rescues white adipose tissue function in vivo.

    Directory of Open Access Journals (Sweden)

    Yuan Gao

    Full Text Available Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis, a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/- mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/- mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/- displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/- mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice.

  1. Allele Compensation in Tip60+/− Mice Rescues White Adipose Tissue Function In Vivo

    Science.gov (United States)

    Gao, Yuan; Hamers, Nicole; Rakhshandehroo, Maryam; Berger, Ruud; Lough, John; Kalkhoven, Eric

    2014-01-01

    Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis), a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/− mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/− mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/− displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/− mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice. PMID:24870614

  2. Consequences of exposure to ionizing radiation for effector T cell function in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Rouse, B.T.; Hartley, D.; Doherty, P.C. (Univ. of Tennessee, Knoxville (USA))

    1989-01-01

    The adoptive transfer of acutely primed and memory virus-immune CD8+ T cells causes enhanced meningitis in both cyclophosphamide (Cy) suppressed, and unsuppressed, recipients infected with lymphocytic choriomeningitis virus (LCMV). The severity of meningitis is assessed by counting cells in cerebrospinal fluid (CSF) obtained from the cisterna magna, which allows measurement of significant inflammatory process ranging from 3 to more than 300 times the background number of cells found in mice injected with virus alone. Exposure of the donor immune population to ionizing radiation prior to transfer has shown that activated T cells from mice primed 7 or 8 days previously with virus may still promote a low level of meningitis in unsuppressed recipients following as much as 800 rads, while this effect is lost totally in Cy-suppressed mice at 600 rads. Memory T cells are more susceptible and show no evidence of in vivo effector function in either recipient population subsequent to 400 rads, a dose level which also greatly reduces the efficacy of acutely-primed T cells. The results are interpreted as indicating that heavily irradiated cells that are already fully functional show evidence of primary localization to the CNS and a limited capacity to cause pathology. Secondary localization, and events that require further proliferation of the T cells in vivo, are greatly inhibited by irradiation.

  3. In Vivo Phosphorylation Site Mapping and Functional Characterization of Arabidopsis Phototropin 1

    Institute of Scientific and Technical Information of China (English)

    Stuart Sullivan; Catriona E. Thomson; Douglas J.Lamont; Matthew A. Jones; John M.Christie

    2008-01-01

    Phototropins (phot1 and phot2) are blue-light receptor kinases controlling a range of responses that optimize the photosynthetic efficiency of plants. Light sensing is mediated by two flavin-binding motifs, known as LOV1 and LOV2,located within the N-terminal region of the protein. Photoexcitation via LOV2 leads to activation of the C-terminal kinase domain and consequently receptor autophosphorylation. However, knowledge of the in-vivo phosphorylation sites for Arabidopsis phototropins is lacking and has impeded progress in elucidating the functional significance of receptor phosphorylation. We have purified phot1 from Arabidopsis and identified the in-vivo sites of receptor phosphorylation by liquid chromatography tandem mass spectrometry. Arabidopsis-derived phot1 binds flavin mononucleotide as chromophore and is phosphorylated at four major sites located upstream of LOV2 (Ser58, Ser85, Ser350, and Ser410), three of which are induced by blue light. Nevertheless, structure-function analysis indicates that the biological activity of phot1 can be attributed to a modular unit comprising the LOV2-kinase region of the protein. Thus, peptide regions upstream of LOV2, including the sites of receptor phosphorylation identified here, do not appear to be important for receptor signaling. By contrast, these regions may be necessary for maximizing stomatal performance and possibly light-induced relocalization of phot1.

  4. In Vivo Functional and Transcriptional Profiling of Bone Marrow Stem Cells after Transplantation into Ischemic Myocardium

    Science.gov (United States)

    Sheikh, Ahmad Y.; Huber, Bruno C.; Narsinh, Kazim H.; Spin, Joshua M.; van der Bogt, Koen; de Almeida, Patricia E.; Ransohoff, Katherine J.; Kraft, Daniel L.; Fajardo, Giovanni; Ardigo, Diego; Ransohoff, Julia; Bernstein, Daniel; Fischbein, Michael P.; Robbins, Robert C.; Wu, Joseph C.

    2011-01-01

    Objective Clinical trials of bone marrow-derived stem cell therapy for the heart have yielded variable results. The basic mechanism(s) that underlie their potential efficacy remains unknown. In the present study, we evaluate the survival kinetics, transcriptional response, and functional outcome of intramyocardial bone marrow mononuclear cell (BMMC) transplantation for cardiac repair in murine myocardial infarction model. Methods and Results We utilized molecular-genetic bioluminescence imaging and high throughput transcriptional profiling to evaluate the in vivo survival kinetics and gene expression changes of transplanted BMMCs after their engraftment into ischemic myocardium. Our results demonstrate short-lived survival of cells following transplant, with less than 1% of cells surviving by 6 weeks post-transplantation. Moreover, transcriptomic analysis of BMMCs revealed non-specific upregulation of various cell regulatory genes with a marked downregulation of cell differentiation and maturation pathways. BMMC therapy caused limited improvement of heart function as assessed by echocardiography, invasive hemodynamics, and positron emission tomography (PET). Histological evaluation of cell fate further confirmed findings of the in vivo cell tracking and transcriptomic analysis. Conclusions Collectively, these data suggest that BMMC therapy, in its present iteration, may be less efficacious than once thought. Additional refinement of existing cell delivery protocols should be considered to induce better therapeutic efficacy. PMID:22034515

  5. Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Dinnyes Andras

    2007-03-01

    Full Text Available Abstract Background Real-time PCR is an efficient tool to measure transcripts and provide valuable quantitative information on gene expression of preimplantation stage embryos. Finding valid reference genes for normalization is essential to interpret the real-time PCR results accurately, and understand the biological dynamics during early development. The use of reference genes also known as housekeeping genes is the most widely applied approach. However, the different genes are not systematically compared, and as a result there is no uniformity between studies in selecting the reference gene. The goals of this study were to compare a wide selection of the most commonly used housekeeping genes in mouse oocytes and preimplantation stage embryos produced under different culture conditions, and select the best stable genes for normalization of gene expression data. Results Quantitative real time PCR method was used to evaluate 12 commonly used housekeeping genes (Actb, Gapdh, H2afz, Hprt, Ppia, Ubc, Eef1e1, Tubb4, Hist2h2aa1, Tbp, Bmp7, Polr2a in multiple individual embryos representing six different developmental stages. The results were analysed, and stable genes were selected using the geNorm software. The expression pattern was almost similar despite differences in the culture system; however, the transcript levels were affected by culture conditions. The genes have showed various stabilities, and have been ranked accordingly. Conclusion Compared to earlier studies with similar objectives, we used a unique approach in analysing larger number of genes, comparing embryo samples derived in vivo or in vitro, analysing the expression in the early and late maternal to zygote transition periods separately, and using multiple individual embryos. Based on detailed quantification, pattern analyses and using the geNorm application, we found Ppia, H2afz and Hprt1 genes to be the most stable across the different stages and culture conditions, while Actb

  6. Value of phagocyte function screening for immunotoxicity of nanoparticles in vivo

    Directory of Open Access Journals (Sweden)

    Fröhlich E

    2015-05-01

    Full Text Available Eleonore Fröhlich Center for Medical Research, Medical University of Graz, Graz, Austria Abstract: Nanoparticles (NPs present in the environment and in consumer products can cause immunotoxic effects. The immune system is very complex, and in vivo studies are the gold standard for evaluation. Due to the increased amount of NPs that are being developed, cellular screening assays to decrease the amount of NPs that have to be tested in vivo are highly needed. Effects on the unspecific immune system, such as effects on phagocytes, might be suitable for screening for immunotoxicity because these cells mediate unspecific and specific immune responses. They are present at epithelial barriers, in the blood, and in almost all organs. This review summarizes the effects of carbon, metal, and metal oxide NPs used in consumer and medical applications (gold, silver, titanium dioxide, silica dioxide, zinc oxide, and carbon nanotubes and polystyrene NPs on the immune system. Effects in animal exposures through different routes are compared to the effects on isolated phagocytes. In addition, general problems in the testing of NPs, such as unknown exposure doses, as well as interference with assays are mentioned. NPs appear to induce a specific immunotoxic pattern consisting of the induction of inflammation in normal animals and aggravation of pathologies in disease models. The evaluation of particle action on several phagocyte functions in vitro may provide an indication on the potency of the particles to induce immunotoxicity in vivo. In combination with information on realistic exposure levels, in vitro studies on phagocytes may provide useful information on the health risks of NPs. Keywords: immunotoxicity, phagocytes, cytokines, respiratory burst, nitric oxide generation, phagocytosis

  7. In vivo, label-free, three-dimensional quantitative imaging of liver surface using multi-photon microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zhuo, Shuangmu, E-mail: shuangmuzhuo@gmail.com, E-mail: hanry-yu@nuhs.edu.sg [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Institute of Laser and Optoelectronics Technology, Fujian Normal University, Fuzhou 350007 (China); Yan, Jie [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, #04-01, 138669 Singapore (Singapore); Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, MD 11 #04-01A, 117599 Singapore (Singapore); Kang, Yuzhan [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Xu, Shuoyu [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, #04-01, 138669 Singapore (Singapore); Computation and System Biology Program, Singapore-MIT Alliance, 4 Engineering Drive 3, E4-04-10, 117576 Singapore (Singapore); Peng, Qiwen [Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, #04-01, 138669 Singapore (Singapore); Computation and System Biology Program, Singapore-MIT Alliance, 4 Engineering Drive 3, E4-04-10, 117576 Singapore (Singapore); Mechanobiology Institute, 5A Engineering Drive 1, T-Lab #05-01, 117411 Singapore (Singapore); and others

    2014-07-14

    Various structural features on the liver surface reflect functional changes in the liver. The visualization of these surface features with molecular specificity is of particular relevance to understanding the physiology and diseases of the liver. Using multi-photon microscopy (MPM), we have developed a label-free, three-dimensional quantitative and sensitive method to visualize various structural features of liver surface in living rat. MPM could quantitatively image the microstructural features of liver surface with respect to the sinuosity of collagen fiber, the elastic fiber structure, the ratio between elastin and collagen, collagen content, and the metabolic state of the hepatocytes that are correlative with the pathophysiologically induced changes in the regions of interest. This study highlights the potential of this technique as a useful tool for pathophysiological studies and possible diagnosis of the liver diseases with further development.

  8. Genome-wide compendium and functional assessment of in vivo heart enhancers.

    Science.gov (United States)

    Dickel, Diane E; Barozzi, Iros; Zhu, Yiwen; Fukuda-Yuzawa, Yoko; Osterwalder, Marco; Mannion, Brandon J; May, Dalit; Spurrell, Cailyn H; Plajzer-Frick, Ingrid; Pickle, Catherine S; Lee, Elizabeth; Garvin, Tyler H; Kato, Momoe; Akiyama, Jennifer A; Afzal, Veena; Lee, Ah Young; Gorkin, David U; Ren, Bing; Rubin, Edward M; Visel, Axel; Pennacchio, Len A

    2016-10-05

    Whole-genome sequencing is identifying growing numbers of non-coding variants in human disease studies, but the lack of accurate functional annotations prevents their interpretation. We describe the genome-wide landscape of distant-acting enhancers active in the developing and adult human heart, an organ whose impairment is a predominant cause of mortality and morbidity. Using integrative analysis of >35 epigenomic data sets from mouse and human pre- and postnatal hearts we created a comprehensive reference of >80,000 putative human heart enhancers. To illustrate the importance of enhancers in the regulation of genes involved in heart disease, we deleted the mouse orthologs of two human enhancers near cardiac myosin genes. In both cases, we observe in vivo expression changes and cardiac phenotypes consistent with human heart disease. Our study provides a comprehensive catalogue of human heart enhancers for use in clinical whole-genome sequencing studies and highlights the importance of enhancers for cardiac function.

  9. Plant-PET Scans: In Vivo Mapping of Xylem and Phloem Functioning.

    Science.gov (United States)

    Hubeau, Michiel; Steppe, Kathy

    2015-10-01

    Medical imaging techniques are rapidly expanding in the field of plant sciences. Positron emission tomography (PET) is advancing as a powerful functional imaging technique to decipher in vivo the function of xylem water flow (with (15)O or (18)F), phloem sugar flow (with (11)C or (18)F), and the importance of their strong coupling. However, much remains to be learned about how water flow and sugar distribution are coordinated in intact plants, both under present and future climate regimes. We propose to use PET analysis of plants (plant-PET) to visualize and generate these missing data about integrated xylem and phloem transport. These insights are crucial to understanding how a given environment will affect plant physiological processes and growth. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. The archetypal R90C CADASIL-NOTCH3 mutation retains NOTCH3 function in vivo.

    Science.gov (United States)

    Monet, Marie; Domenga, Valérie; Lemaire, Barbara; Souilhol, Céline; Langa, Francina; Babinet, Charles; Gridley, Thomas; Tournier-Lasserve, Elisabeth; Cohen-Tannoudji, Michel; Joutel, Anne

    2007-04-15

    Cerebral Autosomal Dominant Arteriopathy with Subcortical infarcts and Leukoencephalopathy (CADASIL) is the most prominent known cause of inherited stroke and vascular dementia in human adult. The disease gene, NOTCH3, encodes a transmembrane receptor primarily expressed in arterial smooth muscle cells (SMC). Pathogenic mutations lead to an odd number of cysteine residues within the NOTCH3 extracellular domain (NOTCH3(ECD)), and are associated with progressive accumulation of NOTCH3(ECD) at the SMC plasma membrane. The murine homolog, Notch3, is dispensable for viability but required post-natally for the elaboration and maintenance of arteries. How CADASIL-associated mutations impact NOTCH3 function remains a fundamental, yet unresolved issue. Particularly, whether NOTCH3(ECD) accumulation may titrate the ligand and inhibit the normal pathway is unknown. Herein, using genetic analyses in the mouse, we assessed the functional significance of an archetypal CADASIL-associated mutation (R90C), in vivo, in brain arteries. We show that transgenic mouse lines expressing either the wild-type human NOTCH3 or the mutant R90C human NOTCH3, at comparable and physiological levels, can rescue the arterial defects of Notch3-/- mice to similar degrees. In vivo assessment of NOTCH3/RBP-Jk activity provides evidence that the mutant NOTCH3 protein exhibits normal level of activity in brain arteries. Remarkably, the mutant NOTCH3 protein remains functional and does not exhibit dominant negative interfering activity, even when NOTCH3(ECD) accumulates. Collectively, these data suggest a model that invokes novel pathogenic roles for the mutant NOTCH3 protein rather than compromised NOTCH3 function as the primary determinant of the CADASIL arteriopathy.

  11. STARS is essential to maintain cardiac development and function in vivo via a SRF pathway.

    Directory of Open Access Journals (Sweden)

    Nelson W Chong

    Full Text Available BACKGROUND: STARS (STriated muscle Activator of Rho Signaling is a sarcomeric protein expressed early in cardiac development that acts as an acute stress sensor for pathological remodeling. However the role of STARS in cardiac development and function is incompletely understood. Here, we investigated the role of STARS in heart development and function in the zebrafish model and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: Expression of zebrafish STARS (zSTARS first occurs in the somites by the 16 somite stage [17 hours post fertilization (hpf]. zSTARS is expressed in both chambers of the heart by 48 hpf, and also in the developing brain, jaw structures and pectoral fins. Morpholino-induced knockdown of zSTARS alters atrial and ventricular dimensions and decreases ventricular fractional shortening (measured by high-speed video microscopy, with pericardial edema and decreased or absent circulation [abnormal cardiac phenotypes in 126/164 (77% of morpholino-injected embryos vs. 0/152 (0% of control morpholino embryos]. Co-injection of zsrf (serum response factor mRNA rescues the cardiac phenotype of zSTARS knockdown, resulting in improved fractional shortening and ventricular end-diastolic dimensions. Ectopic over-expression of STARS in vitro activates the STARS proximal promoter, which contains a conserved SRF site. Chromatin immunoprecipitation demonstrates that SRF binds to this site in vivo and the SRF inhibitor CCG-1423 completely blocks STARS proximal reporter activity in H9c2 cells. CONCLUSIONS/SIGNIFICANCE: This study demonstrates for the first time that STARS deficiency severely disrupts cardiac development and function in vivo and revealed a novel STARS-SRF feed-forward autoregulatory loop that could play an essential role in STARS regulation and cardiac function.

  12. Applications of phosphorescent materials for in-vivo imaging of brain structure and function

    Science.gov (United States)

    Boverman, Gregory; Shi, Xiaolei; Cotero, Victoria E.; Filkins, Robert J.; Srivastava, Alok M.; Lorraine, Peter W.; Neculaes, Vasile B.; Ishaque, A. N.

    2016-03-01

    A number of approaches have been developed for in-vivo imaging of neural function at the time scale of action potentials and at the spatial resolution of individual neurons. Remarkable results have been obtained with optogenetics, although the need for genetic modification is an important limitation of these approaches. Similarly, voltage and ion-sensitive dyes allow for optical imaging of action potentials but toxicity remains a problem. Additionally, optical techniques are often only able to be used up to a limited depth. Our preliminary work has shown that nanoparticles of common phosphorescent materials, believed to be generally non-toxic, specifically lutetium oxide and strontium aluminate, can be utilized for cellular imaging, for tomographic imaging, and that the particles can be designed to adhere to neurons. Additionally, lutetium oxide has been shown to be highly X-ray luminescent, potentially allowing for imaging deep within the brain, if the particles can be targeted properly. In ex vivo experiments, we have shown that the phosphorescence of strontium aluminate particles is significantly affected by electric fields similar in strength to those found in the vicinity of the cellular membrane of a neuron. This phenomenon is consistent with early published reports in the electroluminescence literature, namely the Gudden-Pohl effect. We will show results of the ex vivo imaging and dynamic electrical stimulation experiments. We will also show some preliminary ex vivo cell culture results, and will describe plans for future research, focusing on potential in both cell cultures and in vivo for animal models.

  13. Functional evaluation of ES-somatic cell hybrids in vitro and in vivo.

    Science.gov (United States)

    Sumer, Huseyin; Kim, Kitai; Liu, Jun; Ng, Kitwa; Daley, George Q; Verma, Paul J

    2014-06-01

    Embryonic stem cells (ESCs) have previously been reported to reprogram somatic cells following fusion. The resulting ES-somatic cell hybrids have been shown to adopt the transcriptional profile of ESCs, suggesting that the pluripotent program is dominant. ES-somatic cell hybrids have most characteristics of pluripotent cells in vitro; however, it remains unclear whether the somatic genome is an active partner in the hybrid cells or simply retained predominately as silent cargo. Furthermore, the functional properties of ES-somatic cell hybrids in vivo have been limited to studies on their contribution to teratomas and developing embryos/chimeras. The extent of their pluripotency remains largely unclear. Here we determined that the somatic genome is actively transcribed by generating ES-somatic cell hybrids using Rag2-deficient ESCs fused to autologous wild-type somatic cells. Rag2 expression was detected during in vitro differentiation, suggesting that the somatic genome follows the correct temporal cues during differentiation. Furthermore, ES-somatic cell hybrids maintain their tetraploid state following 4 weeks of differentiation in vivo and are immune tolerated when transferred into matched individuals. The ES-somatic cell hybrids can efficiently differentiate into hematopoietic precursors in both myeloid and lymphoid lineages in vitro, suggesting that the somatic genome is actively transcribed following cell fusion based reprogramming. However, the ES-somatic cell hybrids showed an altered hematopoietic potential following in vitro differentiation and were unable to show hematopoietic engraftment in a mouse model.

  14. An in vivo and in vitro comparison of CYP gene induction in mice using liver slices and quantitative RT-PCR.

    Science.gov (United States)

    Martignoni, Marcella; de Kanter, Ruben; Grossi, Pietro; Saturno, Grazia; Barbaria, Elena; Monshouwer, Mario

    2006-02-01

    The scope of this study was to compare in vitro and in vivo cytochrome P450 (CYP) gene induction in mice, using liver slices as an in vitro model. We have chosen to study mice to be able to better interpret CYP induction during long-term safety studies in this species. Mouse liver slices were incubated with beta-naphthoflavone (betaNF), phenobarbital (PB) or dexamethasone (DEX) for 24 h. In addition, in an in vivo study, mice were treated with the same compounds for three days. The mRNA expression of cyp1a1, cyp1a2, cyp2b10 and cyp3a11, which are important for drug metabolism and inducible by xenobiotics, were investigated in vivo and in vitro by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Both in mouse liver slices and in vivo, betaNF was found to be a potent inducer of cyp1a1 and to a lesser extent of cyp1a2. All three compounds induced cyp2b10 mRNA levels, while the cyp3a11 mRNA level was induced only by DEX. Overall, these data demonstrated a good predictive in vitro-in vivo correlation of CYP induction.

  15. Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs

    Science.gov (United States)

    Céspedes, María Virtudes; Fernández, Yolanda; Unzueta, Ugutz; Mendoza, Rosa; Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejando; Álamo, Patricia; Toledo-Rubio, Verónica; Ferrer-Miralles, Neus; Vázquez, Esther; Schwartz, Simó; Abasolo, Ibane; Corchero, José Luis; Mangues, Ramon; Villaverde, Antonio

    2016-01-01

    In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4+ cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals’ endocrine system. PMID:27775083

  16. Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs.

    Science.gov (United States)

    Céspedes, María Virtudes; Fernández, Yolanda; Unzueta, Ugutz; Mendoza, Rosa; Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejando; Álamo, Patricia; Toledo-Rubio, Verónica; Ferrer-Miralles, Neus; Vázquez, Esther; Schwartz, Simó; Abasolo, Ibane; Corchero, José Luis; Mangues, Ramon; Villaverde, Antonio

    2016-10-24

    In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4(+) cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals' endocrine system.

  17. In vivo labelling of functional ribosomes reveals spatial regulation during starvation in Podospora anserina

    Directory of Open Access Journals (Sweden)

    Silar Philippe

    2000-11-01

    Full Text Available Abstract Background To date, in eukaryotes, ribosomal protein expression is known to be regulated at the transcriptional and/or translational levels. But other forms of regulation may be possible. Results Here, we report the successful tagging of functional ribosomal particles with a S7-GFP chimaeric protein, making it possible to observe in vivo ribosome dynamics in the filamentous fungus Podospora anserina. Microscopic observations revealed a novel kind of ribosomal protein regulation during the passage between cell growth and stationary phases, with a transient accumulation of ribosomal proteins and/or ribosome subunits in the nucleus, possibly the nucleolus, being observed at the beginning of stationary phase. Conclusion Nuclear sequestration can be another level of ribosomal protein regulation in eukaryotic cells.This may contribute to the regulation of cell growth and division.

  18. Modulation of the counts and functions of neutrophils and monocytes under in vivo hyperthermia conditions

    DEFF Research Database (Denmark)

    Kappel, M; Kharazmi, A; Nielsen, H

    1994-01-01

    The present work was designed to examine the effect of in vivo hyperthermia on the cell number and functions of polymorphonuclear leucocytes (PMN) and monocytes in human beings. Eight healthy volunteers were immersed into a waterbath (WI) (water temperature 39.5 degrees C) for 2 h, whereby...... their rectal temperature rose to 39.5 degrees C. On a later day they served as their own controls, being immersed into thermoneutral water (34.5 degrees C) for 2 h. Blood samples were collected before immersion, at body temperatures of 38, 39 and 39.5 degrees C as well as 2 h after water immersion...... of blood was significantly enhanced 2 h after hot WI.(ABSTRACT TRUNCATED AT 250 WORDS)...

  19. Invertebrate lysozymes: Diversity and distribution, molecular mechanism and in vivo function

    Indian Academy of Sciences (India)

    Joris M Van Herreweghe; Chris W Michiels

    2012-06-01

    Lysozymes are antibacterial enzymes widely distributed among organisms. Within the animal kingdom, mainly three major lysozyme types occur. Chicken (c)-type lysozyme and goose (g)-type lysozyme are predominantly, but not exclusively, found in vertebrate animals, while the invertebrate (i)-type lysozyme is typical for invertebrate organisms, and hence its name. Since their discovery in 1975, numerous research articles report on the identification of i-type lysozymes in a variety of invertebrate phyla. This review describes the current knowledge on i-type lysozymes, outlining their distribution, molecular mechanism and in vivo function taking the representative from Venerupis philippinarum (formerly Tapes japonica) (Vp-ilys) as a model. In addition, invertebrate g-type and ch-type (chalaropsis) lysozymes, which have been described in molluscs and nematodes, respectively, are also briefly discussed.

  20. Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo

    DEFF Research Database (Denmark)

    Burgess, Rebecca C; Lisby, Michael; Altmannova, Veronika

    2009-01-01

    Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 "anti-recombinase" restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we...... cytologically characterize Srs2 function in vivo and describe a novel mechanism for regulating the initiation of HR. We find that Srs2 is recruited separately to replication and repair centers and identify the genetic requirements for recruitment. In the absence of Srs2 activity, Rad51 foci accumulate......, and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2...

  1. ASSESSMENT OF IN VIVO MECHANICAL MUSCLE FUNCTION IN PATIENTS WITH OSTEOARTHRITIS (OA) OF THE HIP; RELIABILITY

    DEFF Research Database (Denmark)

    Jensen, Carsten; Overgaard, Søren; Aagaard, Per

    2009-01-01

    -joint strength and power a novel setup. MATERIAL AND METHODS Isokinetic contractions for both knee and hip muscles were performed. Reliability for isometric muscle contractions in vivo was recorded and evaluated by use of within subject variability (CVW-S), Spearman correlation and Limits of Agreement (Lo......INTRODUCTION Muscle function in patients with hip OA is not well-studied. We established a new setup of tests in order to monitor patients before and after surgery with total hip arthroplasty (THA). A test-retest protocol was designed to evaluate the reproducibility of single- and multi......A). Both explosive muscle force characteristics (Rate of force development) and maximal isometric force (MVC) were obtained for the affected (aff) and non-affected (n-aff) leg (only data MVC for affected side in this abstract). 20 patients, (age 55.9 ± 4.8; height 174 ± 8; BMI 27.1 ± 4.5) with unilateral...

  2. In vivo BDNF modulation of adult functional and morphological synaptic plasticity at hippocampal mossy fibers.

    Science.gov (United States)

    Gómez-Palacio-Schjetnan, Andrea; Escobar, Martha L

    2008-11-07

    Brain-derived neurotrophic factor (BDNF) has been proposed as a key regulator and mediator of long-term synaptic modifications related to learning and memory maintenance. Our previous studies show that application of high-frequency stimulation (HFS) sufficient to elicit LTP at the dentate gyrus (DG)-CA3 pathway produces mossy fiber structural modifications 7 days after tetanic stimulation. In the present study, we show that acute intrahippocampal microinfusion of BDNF induces a lasting potentiation of synaptic efficacy in the DG-CA3 projection of anesthetized adult rats. Furthermore, we show that BDNF functional modifications in synaptic efficacy are accompanied by a presynaptic structural long-lasting reorganization at the hippocampal mossy fiber pathway. These findings support the idea that BDNF plays an important role as synaptic messenger of activity-dependent synaptic plasticity in the adult mammalian brain, in vivo.

  3. Quantitative amino acid profiling and stable isotopically labeled amino acid tracer enrichment used for in vivo human systemic and tissue kinetics measurements

    DEFF Research Database (Denmark)

    Bornø, Andreas; van Hall, Gerrit

    2014-01-01

    /ion exchange, derivatized using a phenylisothiocyanate reagent and each amino acid was quantitated with its own stable isotopically labeled internal standard (uniformly labeled-(13)C/(15)N). The method was validated according to general recommendations for chromatographic analytical methods. The calibration...... and plasma were 4.4 and 0.8%, and the interday variability was 3.4% and the recovery was 90.5%, respectively. In conclusion, we have developed and validated a method for quantitative amino acid profiling that meets the requirements for systemic and tissue human in vivo amino acid and protein turnover...

  4. Effects of "in vivo" administration of baclofen on rat renal tubular function.

    Science.gov (United States)

    Donato, Verónica; Pisani, Gerardo Bruno; Trumper, Laura; Monasterolo, Liliana Alicia

    2013-09-05

    The effects of the in vivo administration of baclofen on renal tubular transport and aquaporin-2 (AQP2) expression were evaluated. In conscious animals kept in metabolic cages, baclofen (0.01-1mg/kg, s.c.) induced a dose-dependent increment in the urine flow rate (UFR) and in sodium and potassium excretion, associated with an increased osmolal clearance (Closm), a diminished urine to plasma osmolality ratio (Uosm/Posm) and a decrease in AQP2 expression. The above mentioned baclofen effects on functional parameters were corroborated by using conventional renal clearance techniques. Additionally, this model allowed the detection of a diminution in glucose reabsorption. Some experiments were performed with water-deprived or desmopressin-treated rats kept in metabolic cages. Either water deprivation or desmopressin treatment decreased the UFR and increased the Uosm/Posm. Baclofen did not change the Uosm/Posm or AQP2 expression in desmopressin-treated rats; but it increased the UFR and diminished the Uosm/Posm and AQP2 expression in water-deprived animals. These results indicate that in vivo administration of baclofen promotes alterations in proximal tubular transport, since glucose reabsorption was decreased. The distal tubular function was also affected. The increased Closm indicates an alteration in solute reabsorption at the ascending limb of the Henle's loop. The decreased Uosm/Posm and AQP2 expression in controls and in water-deprived, but not in desmopressin-treated rats, lead us to speculate that some effect of baclofen on endogenous vasopressin availability could be responsible for the impaired urine concentrating ability, more than any disturbance in the responsiveness of the renal cells to the hormone.

  5. In vivo neuronal function of the fragile X mental retardation protein is regulated by phosphorylation.

    Science.gov (United States)

    Coffee, R Lane; Williamson, Ashley J; Adkins, Christopher M; Gray, Marisa C; Page, Terry L; Broadie, Kendal

    2012-02-15

    Fragile X syndrome (FXS), caused by loss of the Fragile X Mental Retardation 1 (FMR1) gene product (FMRP), is the most common heritable cause of intellectual disability and autism spectrum disorders. It has been long hypothesized that the phosphorylation of serine 500 (S500) in human FMRP controls its function as an RNA-binding translational repressor. To test this hypothesis in vivo, we employed neuronally targeted expression of three human FMR1 transgenes, including wild-type (hFMR1), dephosphomimetic (S500A-hFMR1) and phosphomimetic (S500D-hFMR1), in the Drosophila FXS disease model to investigate phosphorylation requirements. At the molecular level, dfmr1 null mutants exhibit elevated brain protein levels due to loss of translational repressor activity. This defect is rescued for an individual target protein and across the population of brain proteins by the phosphomimetic, whereas the dephosphomimetic phenocopies the null condition. At the cellular level, dfmr1 null synapse architecture exhibits increased area, branching and bouton number. The phosphomimetic fully rescues these synaptogenesis defects, whereas the dephosphomimetic provides no rescue. The presence of Futsch-positive (microtubule-associated protein 1B) supernumerary microtubule loops is elevated in dfmr1 null synapses. The human phosphomimetic restores normal Futsch loops, whereas the dephosphomimetic provides no activity. At the behavioral level, dfmr1 null mutants exhibit strongly impaired olfactory associative learning. The human phosphomimetic targeted only to the brain-learning center restores normal learning ability, whereas the dephosphomimetic provides absolutely no rescue. We conclude that human FMRP S500 phosphorylation is necessary for its in vivo function as a neuronal translational repressor and regulator of synaptic architecture, and for the manifestation of FMRP-dependent learning behavior.

  6. An in vivo and in vitro comparison of CYP induction in rat liver and intestine using slices and quantitative RT-PCR.

    Science.gov (United States)

    Martignoni, Marcella; de Kanter, Ruben; Grossi, Pietro; Mahnke, Axel; Saturno, Grazia; Monshouwer, Mario

    2004-12-30

    Xenobiotics, including drugs, can influence cytochrome P450 (CYP) activity by upregulating the transcription of CYP genes. To minimize potential drug interactions, it is important to ascertain whether a compound will be an inducer of CYP enzymes early in the development of new therapeutic agents. In vivo and in vitro studies are reported that demonstrate the use of liver and intestinal slices as an in vitro model to predict potential CYP induction in vivo. Rat liver slices and intestinal slices were incubated, for 24 h and 6 h, respectively, with beta-naphthoflavone (betaNF), phenobarbital (PB) or dexamethasone (DEX). In an in vivo study, rats were treated with the same compounds for 3 days. In vivo and in vitro CYP mRNA levels were measured by using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). In addition, CYP enzyme activities were determined in rat liver slices after 48 h incubation. In both rat liver and intestinal slices, betaNF significantly induced CYP1A1, CYP1A2 and CYP2B1 mRNA levels. PB significantly induced CYP2B1. In liver slices a minor induction of CYP1A1 and CYP3A1 by PB was observed, whereas DEX significantly induced CYP3A1, CYP2B1 and CYP1A2 mRNA levels. The induction profiles (qualitative and quantitative) observed in vivo and in vitro are quite similar. All together, these data demonstrate that liver and intestinal slices are a useful and predictive tool to study CYP induction.

  7. In vivo Echocardiographic Assessment of Left Ventricular Function in Transgenic and Gene-Targeted Mice.

    Science.gov (United States)

    Hoit, B D; Walsh, R A

    1997-05-01

    Manipulation of the mammalian genome with transgenic and gene-targeting techniques is a powerful method for unambiguously identifying the molecular mechanisms underlying cardiac development and function. Although the small size of the mouse heart and the rapid heart rates encountered have limited echocardiographic assessment of the murine heart in the past, the use of sophisticated transducers operating at a high frequency results in highly reliable and reproducible image quality. M-mode echocardiography has been shown to provide a good correlation with gravimetrically determined left ventricular mass (LV) and to estimate accurately LV dimensions and systolic function. Doppler interrogation of transvalvular flows permits assessment of global LV systolic and diastolic function independent of ventricular geometry. Linear stress-shortening relations can be determined in the adult mouse with the use of pharmacologically induced changes in systemic arterial pressure, and these relations are capable of detecting changes in myocardial contractility in vivo, relatively independent of loading conditions. The present review focuses on the current advantages and limitations of M-mode and Doppler echocardiography to evaluate cardiac function in mice. (Trends Cardiovasc Med 1997;7:129-134). © 1997, Elsevier Science Inc.

  8. Leptin controls rabbit ovarian function in vivo and in vitro: possible interrelationships with ghrelin.

    Science.gov (United States)

    Sirotkin, A V; Rafay, J; Kotwica, J

    2009-10-01

    The aim of these in vivo and in vitro studies was to examine the role of leptin in the control of plasma hormone concentrations, reproduction, and secretory activity of ovarian granulosa cells. In in vivo experiments, 15 female European domestic rabbit (Oryctolagus cuniculus) were treated with leptin (5 microg animal(-1)d(-1) for 1 wk before induction of ovulation with 25 IU equine chorionic gonadotropin and 0.25 IU human chorionic gonadotropin), and 15 females constituted the control group (treated with phosphate-buffered saline). Plasma concentrations of progesterone (P(4)), testosterone (T), estradiol (E(2)), estrone sulfate (ES), and insulin-like growth factor I (IGF-I) were determined at the estimated day of ovulation by radioimmunoassay (RIA), and number, viability, and body weight of newborns were recorded at parturition. In in vitro experiments, granulosa cells were isolated from periovulatory ovarian follicles of five control and five females treated with ghrelin (10 microg animal(-1)d(-1) for 1 wk before induced ovulation). Isolated cells were cultured for 2 d with and without leptin (0, 1, 10, or 100 ng/mL medium). Secretion of P(4), T, E(2), IGF-I, and prostaglandin F (PGF) was assessed in culture medium by RIA. In in vivo experiments, leptin administrations reduced plasma P(4), T, E(2), ES, and IGF-I levels. Leptin treatments did not affect ovarian weight or total number and body mass of newborns, but the proportion of pregnant females and number of live newborns were significantly higher in leptin-treated females than that in control females. In in vitro experiments, leptin significantly decreased (at 1 and 10 ng/mL) or increased (at 100 ng/mL) P(4) secretion, promoted E(2) and IGF-I (both at 100 ng/mL) secretion, and reduced T (at 1 and 10 ng/mL) and PGF (at 10 ng/mL) secretion. Granulosa cells from ghrelin-treated animals secreted less P(4), T, E(2), and PGF, but not IGF-I, than that secreted by granulosa cells from control animals. Furthermore

  9. Exploring Functional β-Cell Heterogeneity In Vivo Using PSA-NCAM as a Specific Marker

    Science.gov (United States)

    Karaca, Melis; Castel, Julien; Tourrel-Cuzin, Cécile; Brun, Manuel; Géant, Anne; Dubois, Mathilde; Catesson, Sandra; Rodriguez, Marianne; Luquet, Serge; Cattan, Pierre; Lockhart, Brian; Lang, Jochen; Ktorza, Alain

    2009-01-01

    Background The mass of pancreatic β-cells varies according to increases in insulin demand. It is hypothesized that functionally heterogeneous β-cell subpopulations take part in this process. Here we characterized two functionally distinct groups of β-cells and investigated their physiological relevance in increased insulin demand conditions in rats. Methods Two rat β-cell populations were sorted by FACS according to their PSA-NCAM surface expression, i.e. βhigh and βlow-cells. Insulin release, Ca2+ movements, ATP and cAMP contents in response to various secretagogues were analyzed. Gene expression profiles and exocytosis machinery were also investigated. In a second part, βhigh and βlow-cell distribution and functionality were investigated in animal models with decreased or increased β-cell function: the Zucker Diabetic Fatty rat and the 48 h glucose-infused rat. Results We show that β-cells are heterogeneous for PSA-NCAM in rat pancreas. Unlike βlow-cells, βhigh-cells express functional β-cell markers and are highly responsive to various insulin secretagogues. Whereas βlow-cells represent the main population in diabetic pancreas, an increase in βhigh-cells is associated with gain of function that follows sustained glucose overload. Conclusion Our data show that a functional heterogeneity of β-cells, assessed by PSA-NCAM surface expression, exists in vivo. These findings pinpoint new target populations involved in endocrine pancreas plasticity and in β-cell defects in type 2 diabetes. PMID:19440374

  10. Exploring functional beta-cell heterogeneity in vivo using PSA-NCAM as a specific marker.

    Directory of Open Access Journals (Sweden)

    Melis Karaca

    Full Text Available BACKGROUND: The mass of pancreatic beta-cells varies according to increases in insulin demand. It is hypothesized that functionally heterogeneous beta-cell subpopulations take part in this process. Here we characterized two functionally distinct groups of beta-cells and investigated their physiological relevance in increased insulin demand conditions in rats. METHODS: Two rat beta-cell populations were sorted by FACS according to their PSA-NCAM surface expression, i.e. beta(high and beta(low-cells. Insulin release, Ca(2+ movements, ATP and cAMP contents in response to various secretagogues were analyzed. Gene expression profiles and exocytosis machinery were also investigated. In a second part, beta(high and beta(low-cell distribution and functionality were investigated in animal models with decreased or increased beta-cell function: the Zucker Diabetic Fatty rat and the 48 h glucose-infused rat. RESULTS: We show that beta-cells are heterogeneous for PSA-NCAM in rat pancreas. Unlike beta(low-cells, beta(high-cells express functional beta-cell markers and are highly responsive to various insulin secretagogues. Whereas beta(low-cells represent the main population in diabetic pancreas, an increase in beta(high-cells is associated with gain of function that follows sustained glucose overload. CONCLUSION: Our data show that a functional heterogeneity of beta-cells, assessed by PSA-NCAM surface expression, exists in vivo. These findings pinpoint new target populations involved in endocrine pancreas plasticity and in beta-cell defects in type 2 diabetes.

  11. In vitro–in vivo studies of the quantitative effect of calcium, multivitamins and milk on single dose ciprofloxacin bioavailability

    Directory of Open Access Journals (Sweden)

    Baishakhi Dey

    2015-12-01

    Full Text Available Ciprofloxacin, commonly used in India as an anti-microbial for prolonged use in chronic and non-specific indications, may affect the bioavailability of the drug. The drug prescribed is commonly taken with multivitamins, calcium and milk. A simple and reliable analytical methodology obtaining a correlation with in vivo urinary excretion studies using UV and HPLC and in vitro dissolution studies (IVIVC has shown a significant increase in elimination rate of ciprofloxacin co-administered with multivitamins, calcium and milk. Appreciable IVIVC results proved that dissolution studies could serve as an alternative to in vivo bioavailability and also support bio-waivers.

  12. In vitro-in vivo studies of the quantitative effect of calcium, multivitamins and milk on single dose ciprofloxacin bioavailability

    Institute of Scientific and Technical Information of China (English)

    Baishakhi Dey; Prakash Katakam; Fathi H. Assaleh; Babu Rao Chandu; Shanta Kumari Adiki; Analava Mitra

    2015-01-01

    Ciprofloxacin, commonly used in India as an anti-microbial for prolonged use in chronic and non-specific indications, may affect the bioavailability of the drug. The drug prescribed is commonly taken with multivitamins, calcium and milk. A simple and reliable analytical methodology obtaining a correlation with in vivo urinary excretion studies using UV and HPLC and in vitro dissolution studies (IVIVC) has shown a significant increase in elimination rate of ciprofloxacin co-administered with multivitamins, calcium and milk. Appreciable IVIVC results proved that dissolution studies could serve as an alternative to in vivo bioavailability and also support bio-waivers.

  13. Functional significance of glutamate-cysteine ligase modifier for erythrocyte survival in vitro and in vivo.

    Science.gov (United States)

    Föller, M; Harris, I S; Elia, A; John, R; Lang, F; Kavanagh, T J; Mak, T W

    2013-10-01

    Erythrocytes endure constant exposure to oxidative stress. The major oxidative stress scavenger in erythrocytes is glutathione. The rate-limiting enzyme for glutathione synthesis is glutamate-cysteine ligase, which consists of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Here, we examined erythrocyte survival in GCLM-deficient (gclm(-/-)) mice. Erythrocytes from gclm(-/-) mice showed greatly reduced intracellular glutathione. Prolonged incubation resulted in complete lysis of gclm(-/-) erythrocytes, which could be reversed by exogenous delivery of the antioxidant Trolox. To test the importance of GCLM in vivo, mice were treated with phenylhydrazine (PHZ; 0.07 mg/g b.w.) to induce oxidative stress. Gclm(-/-) mice showed dramatically increased hemolysis compared with gclm(+/+) controls. In addition, PHZ-treated gclm(-/-) mice displayed markedly larger accumulations of injured erythrocytes in the spleen than gclm(+/+) mice within 24 h of treatment. Iron staining indicated precipitations of the erythrocyte-derived pigment hemosiderin in kidney tubules of gclm(-/-) mice and none in gclm(+/+) controls. In fact, 24 h after treatment, kidney function began to diminish in gclm(-/-) mice as evident from increased serum creatinine and urea. Consequently, while all PHZ-treated gclm(+/+) mice survived, 90% of PHZ-treated gclm(-/-) mice died within 5 days of treatment. In vitro, upon incubation in the absence or presence of additional oxidative stress, gclm(-/-) erythrocytes exposed significantly more phosphatidylserine, a cell death marker, than gclm(+/+) erythrocytes, an effect at least partially due to increased cytosolic Ca(2+) concentration. Under resting conditions, gclm(-/-) mice exhibited reticulocytosis, indicating that the enhanced erythrocyte death was offset by accelerated erythrocyte generation. GCLM is thus indispensable for erythrocyte survival, in vitro and in vivo, during oxidative stress.

  14. In vivo optogenetic tracing of functional corticocortical connections between motor forelimb areas

    Directory of Open Access Journals (Sweden)

    Riichiro eHira

    2013-04-01

    Full Text Available Interactions between distinct motor cortical areas are essential for coordinated motor behaviors. In rodents, the motor cortical forelimb areas are divided into at least two distinct areas: the rostral forelimb area (RFA and the caudal forelimb area (CFA. The RFA is thought to be an equivalent of the premotor cortex in primates, whereas the CFA is believed to be an equivalent of the primary motor cortex. Although reciprocal connections between the RFA and the CFA have been anatomically identified in rats, it is unknown whether there are functional connections between these areas that can induce postsynaptic spikes. In this study, we used an in vivo Channelrhodopsin-2 photostimulation method to trace the functional connections between the mouse RFA and CFA. Simultaneous electrical recordings were utilized to detect spiking activities induced by synaptic inputs originating from photostimulated areas. This method, in combination with anatomical tracing, demonstrated that the RFA receives strong functional projections from layer 2/3 and/or layer 5a, but not from layer 5b, of the CFA. Further, the CFA receives strong projections from layer 5b neurons of the RFA. The onset latency of electrical responses evoked in remote areas upon photostimulation of the other areas was approximately 10 ms, which is consistent with the synaptic connectivity between these areas. Our results suggest that neuronal activities in the RFA and the CFA during movements are formed through asymmetric reciprocal connections.

  15. Atypical Membrane Topology and Heteromeric Function of Drosophila Odorant Receptors In Vivo.

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available Drosophila olfactory sensory neurons (OSNs each express two odorant receptors (ORs: a divergent member of the OR family and the highly conserved, broadly expressed receptor OR83b. OR83b is essential for olfaction in vivo and enhances OR function in vitro, but the molecular mechanism by which it acts is unknown. Here we demonstrate that OR83b heterodimerizes with conventional ORs early in the endomembrane system in OSNs, couples these complexes to the conserved ciliary trafficking pathway, and is essential to maintain the OR/OR83b complex within the sensory cilia, where odor signal transduction occurs. The OR/OR83b complex is necessary and sufficient to promote functional reconstitution of odor-evoked signaling in sensory neurons that normally respond only to carbon dioxide. Unexpectedly, unlike all known vertebrate and nematode chemosensory receptors, we find that Drosophila ORs and OR83b adopt a novel membrane topology with their N-termini and the most conserved loops in the cytoplasm. These loops mediate direct association of ORs with OR83b. Our results reveal that OR83b is a universal and integral part of the functional OR in Drosophila. This atypical heteromeric and topological design appears to be an insect-specific solution for odor recognition, making the OR/OR83b complex an attractive target for the development of highly selective insect repellents to disrupt olfactory-mediated host-seeking behaviors of insect disease vectors.

  16. Contrast-enhanced optical coherence tomography with picomolar sensitivity for functional in vivo imaging

    Science.gov (United States)

    Liba, Orly; Sorelle, Elliott D.; Sen, Debasish; de La Zerda, Adam

    2016-03-01

    Optical Coherence Tomography (OCT) enables real-time imaging of living tissues at cell-scale resolution over millimeters in three dimensions. Despite these advantages, functional biological studies with OCT have been limited by a lack of exogenous contrast agents that can be distinguished from tissue. Here we report an approach to functional OCT imaging that implements custom algorithms to spectrally identify unique contrast agents: large gold nanorods (LGNRs). LGNRs exhibit 110-fold greater spectral signal per particle than conventional GNRs, which enables detection of individual LGNRs in water and concentrations as low as 250 pM in the circulation of living mice. This translates to ~40 particles per imaging voxel in vivo. Unlike previous implementations of OCT spectral detection, the methods described herein adaptively compensate for depth and processing artifacts on a per sample basis. Collectively, these methods enable high-quality noninvasive contrast-enhanced imaging of OCT in living subjects, including detection of tumor microvasculature at twice the depth achievable with conventional OCT. Additionally, multiplexed detection of spectrally-distinct LGNRs was demonstrated to observe discrete patterns of lymphatic drainage and identify individual lymphangions and lymphatic valve functional states. These capabilities provide a powerful platform for molecular imaging and characterization of tissue noninvasively at cellular resolution, called MOZART.

  17. How Energy Metabolism Supports Cerebral Function: Insights from (13)C Magnetic Resonance Studies In vivo.

    Science.gov (United States)

    Sonnay, Sarah; Gruetter, Rolf; Duarte, João M N

    2017-01-01

    Cerebral function is associated with exceptionally high metabolic activity, and requires continuous supply of oxygen and nutrients from the blood stream. Since the mid-twentieth century the idea that brain energy metabolism is coupled to neuronal activity has emerged, and a number of studies supported this hypothesis. Moreover, brain energy metabolism was demonstrated to be compartmentalized in neurons and astrocytes, and astrocytic glycolysis was proposed to serve the energetic demands of glutamatergic activity. Shedding light on the role of astrocytes in brain metabolism, the earlier picture of astrocytes being restricted to a scaffold-associated function in the brain is now out of date. With the development and optimization of non-invasive techniques, such as nuclear magnetic resonance spectroscopy (MRS), several groups have worked on assessing cerebral metabolism in vivo. In this context, (1)H MRS has allowed the measurements of energy metabolism-related compounds, whose concentrations can vary under different brain activation states. (1)H-[(13)C] MRS, i.e., indirect detection of signals from (13)C-coupled (1)H, together with infusion of (13)C-enriched glucose has provided insights into the coupling between neurotransmission and glucose oxidation. Although these techniques tackle the coupling between neuronal activity and metabolism, they lack chemical specificity and fail in providing information on neuronal and glial metabolic pathways underlying those processes. Currently, the improvement of detection modalities (i.e., direct detection of (13)C isotopomers), the progress in building adequate mathematical models along with the increase in magnetic field strength now available render possible detailed compartmentalized metabolic flux characterization. In particular, direct (13)C MRS offers more detailed dataset acquisitions and provides information on metabolic interactions between neurons and astrocytes, and their role in supporting neurotransmission. Here

  18. How Energy Metabolism Supports Cerebral Function: Insights from 13C Magnetic Resonance Studies In vivo

    Directory of Open Access Journals (Sweden)

    Sarah Sonnay

    2017-05-01

    Full Text Available Cerebral function is associated with exceptionally high metabolic activity, and requires continuous supply of oxygen and nutrients from the blood stream. Since the mid-twentieth century the idea that brain energy metabolism is coupled to neuronal activity has emerged, and a number of studies supported this hypothesis. Moreover, brain energy metabolism was demonstrated to be compartmentalized in neurons and astrocytes, and astrocytic glycolysis was proposed to serve the energetic demands of glutamatergic activity. Shedding light on the role of astrocytes in brain metabolism, the earlier picture of astrocytes being restricted to a scaffold-associated function in the brain is now out of date. With the development and optimization of non-invasive techniques, such as nuclear magnetic resonance spectroscopy (MRS, several groups have worked on assessing cerebral metabolism in vivo. In this context, 1H MRS has allowed the measurements of energy metabolism-related compounds, whose concentrations can vary under different brain activation states. 1H-[13C] MRS, i.e., indirect detection of signals from 13C-coupled 1H, together with infusion of 13C-enriched glucose has provided insights into the coupling between neurotransmission and glucose oxidation. Although these techniques tackle the coupling between neuronal activity and metabolism, they lack chemical specificity and fail in providing information on neuronal and glial metabolic pathways underlying those processes. Currently, the improvement of detection modalities (i.e., direct detection of 13C isotopomers, the progress in building adequate mathematical models along with the increase in magnetic field strength now available render possible detailed compartmentalized metabolic flux characterization. In particular, direct 13C MRS offers more detailed dataset acquisitions and provides information on metabolic interactions between neurons and astrocytes, and their role in supporting neurotransmission. Here

  19. Nature, source and function of pigments in tardigrades: in vivo raman imaging of carotenoids in Echiniscus blumi.

    Science.gov (United States)

    Bonifacio, Alois; Guidetti, Roberto; Altiero, Tiziana; Sergo, Valter; Rebecchi, Lorena

    2012-01-01

    Tardigrades are microscopic aquatic animals with remarkable abilities to withstand harsh physical conditions such as dehydration or exposure to harmful highly energetic radiation. The mechanisms responsible for such robustness are presently little known, but protection against oxidative stresses is thought to play a role. Despite the fact that many tardigrade species are variously pigmented, scarce information is available about this characteristic. By applying Raman micro-spectroscopy on living specimens, pigments in the tardigrade Echiniscus blumi are identified as carotenoids, and their distribution within the animal body is visualized. The dietary origin of these pigments is demonstrated, as well as their presence in the eggs and in eye-spots of these animals, together with their absence in the outer layer of the animal (i.e., cuticle and epidermis). Using in-vivo semi-quantitative Raman micro-spectroscopy, a decrease in carotenoid content is detected after inducing oxidative stress, demonstrating that this approach can be used for studying the role of carotenoids in oxidative stress-related processes in tardigrades. This approach could be thus used in further investigations to test several hypotheses concerning the function of these carotenoids in tardigrades as photo-protective pigments against ionizing radiations or as antioxidants defending these organisms against the oxidative stress occurring during desiccation processes.

  20. Nature, source and function of pigments in tardigrades: in vivo raman imaging of carotenoids in Echiniscus blumi.

    Directory of Open Access Journals (Sweden)

    Alois Bonifacio

    Full Text Available Tardigrades are microscopic aquatic animals with remarkable abilities to withstand harsh physical conditions such as dehydration or exposure to harmful highly energetic radiation. The mechanisms responsible for such robustness are presently little known, but protection against oxidative stresses is thought to play a role. Despite the fact that many tardigrade species are variously pigmented, scarce information is available about this characteristic. By applying Raman micro-spectroscopy on living specimens, pigments in the tardigrade Echiniscus blumi are identified as carotenoids, and their distribution within the animal body is visualized. The dietary origin of these pigments is demonstrated, as well as their presence in the eggs and in eye-spots of these animals, together with their absence in the outer layer of the animal (i.e., cuticle and epidermis. Using in-vivo semi-quantitative Raman micro-spectroscopy, a decrease in carotenoid content is detected after inducing oxidative stress, demonstrating that this approach can be used for studying the role of carotenoids in oxidative stress-related processes in tardigrades. This approach could be thus used in further investigations to test several hypotheses concerning the function of these carotenoids in tardigrades as photo-protective pigments against ionizing radiations or as antioxidants defending these organisms against the oxidative stress occurring during desiccation processes.

  1. REGRESSION OF WHITE SPOT ENAMEL LESIONS - A NEW OPTICAL METHOD FOR QUANTITATIVE LONGITUDINAL EVALUATION IN-VIVO

    NARCIS (Netherlands)

    OGAARD, B; TENBOSCH, JJ

    This article describes a new nondestructive optical method for evaluation of lesion regression in vivo. White spot caries lesions were induced with orthodontic bands in two vital premolars of seven patients. The teeth were banded for 4 weeks with special orthodontic bands that allowed plaque

  2. REGRESSION OF WHITE SPOT ENAMEL LESIONS - A NEW OPTICAL METHOD FOR QUANTITATIVE LONGITUDINAL EVALUATION IN-VIVO

    NARCIS (Netherlands)

    OGAARD, B; TENBOSCH, JJ

    1994-01-01

    This article describes a new nondestructive optical method for evaluation of lesion regression in vivo. White spot caries lesions were induced with orthodontic bands in two vital premolars of seven patients. The teeth were banded for 4 weeks with special orthodontic bands that allowed plaque accumul

  3. Quantitative Comparison of 21 Protocols for Labeling Hippocampal Subfields and Parahippocampal Cortical Subregions in In Vivo MRI: Towards Developing a Harmonized Segmentation Protocol

    DEFF Research Database (Denmark)

    Yushkevich, Paul A.; Amaral, Robert S.C.; Augustinack, Jean C.

    2015-01-01

    Objective: An increasing number of human in vivo magnetic resonance imaging (MRI) studies have focused on examining the structure and function of the subfields of the hippocampal formation (the dentate gyrus, CA fields 1 − 3, and the subiculum) and subregions of the parahippocampal gyrus...

  4. Quantitative evaluation of statistical inference in resting state functional MRI.

    Science.gov (United States)

    Yang, Xue; Kang, Hakmook; Newton, Allen; Landman, Bennett A

    2012-01-01

    Modern statistical inference techniques may be able to improve the sensitivity and specificity of resting state functional MRI (rs-fMRI) connectivity analysis through more realistic characterization of distributional assumptions. In simulation, the advantages of such modern methods are readily demonstrable. However quantitative empirical validation remains elusive in vivo as the true connectivity patterns are unknown and noise/artifact distributions are challenging to characterize with high fidelity. Recent innovations in capturing finite sample behavior of asymptotically consistent estimators (i.e., SIMulation and EXtrapolation - SIMEX) have enabled direct estimation of bias given single datasets. Herein, we leverage the theoretical core of SIMEX to study the properties of inference methods in the face of diminishing data (in contrast to increasing noise). The stability of inference methods with respect to synthetic loss of empirical data (defined as resilience) is used to quantify the empirical performance of one inference method relative to another. We illustrate this new approach in a comparison of ordinary and robust inference methods with rs-fMRI.

  5. In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin.

    Science.gov (United States)

    Lark, Arianna R; Kitamoto, Toshihiro; Martin, Jean-René

    2016-01-08

    Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca(2+)-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and - with the addition of its cofactor coelenterazine - emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca(2+)-transients and Ca(2+)-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca(2+)-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

  6. A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen.

    Science.gov (United States)

    Tikhonova, Elena B; Ethayathulla, Abdul S; Su, Yue; Hariharan, Parameswaran; Xie, Shicong; Guan, Lan

    2015-01-28

    A highly diverse DNA library coding for ankyrin seven-repeat proteins (ANK-N5C) was designed and constructed by a PCR-based combinatorial assembly strategy. A bacterial melibiose fermentation assay was adapted for in vivo functional screen. We isolated a transcription blocker that completely inhibits the melibiose-dependent expression of α-galactosidase (MelA) and melibiose permease (MelB) of Escherichia coli by specifically preventing activation of the melAB operon. High-resolution crystal structural determination reveals that the designed ANK-N5C protein has a typical ankyrin fold, and the specific transcription blocker, ANK-N5C-281, forms a domain-swapped dimer. Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein. All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins. Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents.

  7. In vivo endoscopic tissue diagnostics based on spectroscopic absorption, scattering, and phase function properties.

    Science.gov (United States)

    Thueler, Philippe; Charvet, Igor; Bevilacqua, Frederic; St Ghislain, M; Ory, G; Marquet, Pierre; Meda, Paolo; Vermeulen, Ben; Depeursinge, Christian

    2003-07-01

    A fast spectroscopic system for superficial and local determination of the absorption and scattering properties of tissue (480 to 950 nm) is described. The probe can be used in the working channel of an endoscope. The scattering properties include the reduced scattering coefficient and a parameter of the phase function called gamma, which depends on its first two moments. The inverse problem algorithm is based on the fit of absolute reflectance measurements to cubic B-spline functions derived from the interpolation of a set of Monte Carlo simulations. The algorithm's robustness was tested with simulations altered with various amounts of noise. The method was also assessed on tissue phantoms of known optical properties. Finally, clinical measurements performed endoscopically in vivo in the stomach of human subjects are presented. The absorption and scattering properties were found to be significantly different in the antrum and in the fundus and are correlated with histopathologic observations. The method and the instrument show promise for noninvasive tissue diagnostics of various epithelia.

  8. Caspase inhibitors promote vestibular hair cell survival and function after aminoglycoside treatment in vivo

    Science.gov (United States)

    Matsui, Jonathan I.; Haque, Asim; Huss, David; Messana, Elizabeth P.; Alosi, Julie A.; Roberson, David W.; Cotanche, Douglas A.; Dickman, J. David; Warchol, Mark E.

    2003-01-01

    The sensory hair cells of the inner ear undergo apoptosis after acoustic trauma or aminoglycoside antibiotic treatment, causing permanent auditory and vestibular deficits in humans. Previous studies have demonstrated a role for caspase activation in hair cell death and ototoxic injury that can be reduced by concurrent treatment with caspase inhibitors in vitro. In this study, we examined the protective effects of caspase inhibition on hair cell death in vivo after systemic injections of aminoglycosides. In one series of experiments, chickens were implanted with osmotic pumps that administrated the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD) into inner ear fluids. One day after the surgery, the animals received a 5 d course of treatment with streptomycin, a vestibulotoxic aminoglycoside. Direct infusion of zVAD into the vestibule significantly increased hair cell survival after streptomycin treatment. A second series of experiments determined whether rescued hair cells could function as sensory receptors. Animals treated with streptomycin displayed vestibular system impairment as measured by a greatly reduced vestibulo-ocular response (VOR). In contrast, animals that received concurrent systemic administration of zVAD with streptomycin had both significantly greater hair cell survival and significantly increased VOR responses, as compared with animals treated with streptomycin alone. These findings suggest that inhibiting the activation of caspases promotes the survival of hair cells and protects against vestibular function deficits after aminoglycoside treatment.

  9. Topical Formulation Containing Beeswax-Based Nanoparticles Improved In Vivo Skin Barrier Function.

    Science.gov (United States)

    Souza, Carla; de Freitas, Luis Alexandre Pedro; Maia Campos, Patrícia Maria Berardo Gonçalves

    2017-02-17

    Lipid nanoparticles have shown many advantages for treatment/prevention of skin disorders with damaged skin barrier function. Beeswax is a favorable candidate for the development of nanosystems in the cosmetic and dermatological fields because of its advantages for the development of products for topical application. In the present study, beeswax-based nanoparticles (BNs) were prepared using the hot melt microemulsion technique and incorporated to a gel-cream formulation. The formulation was subsequently evaluated for its rheological stability and effect on stratum corneum water content (SCWC) and transepidermal water loss (TEWL) using in vivo biophysical techniques. BNs resulted in mean particle size of 95.72 ± 9.63 nm and zeta potential of -9.85 ± 0.57 mV. BN-loaded formulation showed shear thinning behavior, well adjusted by the Herschel-Bulkley model, and a small thixotropy index that were stable for 28 days at different temperatures. BN-loaded formulation was also able to simultaneously decrease the TEWL and increase the SCWC values 28 days after treatment. In conclusion, the novel beeswax-based nanoparticles showed potential for barrier recovery and open the perspective for its commercial use as a novel natural active as yet unexplored in the field of dermatology and cosmetics for treatment of skin diseases with damaged skin barrier function.

  10. Quantitative analysis of sodium fast and slow component in in vivo human brain tissue using MR Na image

    Energy Technology Data Exchange (ETDEWEB)

    Hirai, Hirokazu; Yamasaki, Katsuhito; Kidena, Hitoshi; Kono, Michio (Kobe Univ. (Japan). School of Medicine)

    1992-12-01

    In vivo sodium concentrations in the normal brain tissue and a tumorous tissue were analyzed using MR Na image. The nuclear magnetic resonance enabled us to divide the signal from sodium in the living tissue into 2 parts based on the differences of T[sub 2] value. Those are fast component having the T[sub 2] value of less than 5 msec and slow component of 15-40 msec. We investigated the effect of macromolecules on T[sub 2] value of sodium image using polyvinylalcohol (PVA) powder. MR Na image was taken with the parameters of TR/TD, 110 ms/1.9 ms (FID image) and TR/TE, 110 ms/20 ms (SE image). Saline solution showed high intensity on both FID image and SE image. Saline solution added PVA (PVA phantom) also showed high intensity on FID image, whereas the signal intensity of PVA phantom in SE image extinguished. To know the relation between the signal intensity and sodium concentration, sodium concentration-signal intensity curve was obtained using phantoms with various sodium concentrations (0.05-1.0%). This curve showed a direct proportion between sodium concentration and signal intensity on Na image. We measured further the sodium concentrations of the human brain tissue. Sodium phantoms were arranged around the heads and the MR Na images of the normal brains from 3 volunteers and a patient with a brain tumor (meningioma) were taken. The sodium concentrations of occipital lobe, basal ganglia and the tumorous tissue were calculated using the sodium concentration-signal intensity curve obtained from the phantoms arranged around the heads. Two tailed t-test shows significant differences (p<0.01) in total sodium and slow component between occipital lobe and basal ganglia. Further more high concentration of fast component in tumorous tissue was observed. As fast component reflects the intracellular condition, present experiments suggest that measurement of fast component may be useful for obtaining the functional information of the brain tissue. (author).

  11. Conditional gene deletion reveals functional redundancy of GABAB receptors in peripheral nociceptors in vivo

    Directory of Open Access Journals (Sweden)

    Bettler Bernhard

    2009-11-01

    changed upon a specific deletion of GABAB receptors from peripheral nociceptive neurons in vivo. This lets us conclude that GABAB receptors in the peripheral nervous system play a less important role than those in the central nervous system in the regulation of pain.

  12. In vivo mapping of the functional regions of the DEAD-box helicase Vasa

    Directory of Open Access Journals (Sweden)

    Mehrnoush Dehghani

    2015-03-01

    Full Text Available The maternally expressed Drosophila melanogaster DEAD-box helicase Vasa (Vas is necessary for many cellular and developmental processes, including specification of primordial germ cells (pole cells, posterior patterning of the embryo, piRNA-mediated repression of transposon-encoded mRNAs, translational activation of gurken (grk mRNA, and completion of oogenesis itself. Vas protein accumulates in the perinuclear nuage in nurse cells soon after their specification, and then at stage 10 Vas translocates to the posterior pole plasm of the oocyte. We produced a series of transgenic constructs encoding eGFP-Vas proteins carrying mutations affecting different regions of the protein, and analyzed in vivo which Vas functions each could support. We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification. One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases. Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression. We conclude from these experiments that Vas, a multifunctional protein, uses different domains and different molecular associations to carry out its various cellular and developmental roles.

  13. Soil engineering in vivo: harnessing natural biogeochemical systems for sustainable, multi-functional engineering solutions.

    Science.gov (United States)

    DeJong, Jason T; Soga, Kenichi; Banwart, Steven A; Whalley, W Richard; Ginn, Timothy R; Nelson, Douglas C; Mortensen, Brina M; Martinez, Brian C; Barkouki, Tammer

    2011-01-06

    Carbon sequestration, infrastructure rehabilitation, brownfields clean-up, hazardous waste disposal, water resources protection and global warming-these twenty-first century challenges can neither be solved by the high-energy consumptive practices that hallmark industry today, nor by minor tweaking or optimization of these processes. A more radical, holistic approach is required to develop the sustainable solutions society needs. Most of the above challenges occur within, are supported on, are enabled by or grown from soil. Soil, contrary to conventional civil engineering thought, is a living system host to multiple simultaneous processes. It is proposed herein that 'soil engineering in vivo', wherein the natural capacity of soil as a living ecosystem is used to provide multiple solutions simultaneously, may provide new, innovative, sustainable solutions to some of these great challenges of the twenty-first century. This requires a multi-disciplinary perspective that embraces the science of biology, chemistry and physics and applies this knowledge to provide multi-functional civil and environmental engineering designs for the soil environment. For example, can native soil bacterial species moderate the carbonate cycle in soils to simultaneously solidify liquefiable soil, immobilize reactive heavy metals and sequester carbon-effectively providing civil engineering functionality while clarifying the ground water and removing carbon from the atmosphere? Exploration of these ideas has begun in earnest in recent years. This paper explores the potential, challenges and opportunities of this new field, and highlights one biogeochemical function of soil that has shown promise and is developing rapidly as a new technology. The example is used to propose a generalized approach in which the potential of this new field can be fully realized.

  14. Balanced Hydroxyethylstarch (HES 130/0.4 Impairs Kidney Function In-Vivo without Inflammation.

    Directory of Open Access Journals (Sweden)

    Martin Alexander Schick

    Full Text Available Volume therapy is a standard procedure in daily perioperative care, and there is an ongoing discussion about the benefits of colloid resuscitation with hydroxyethylstarch (HES. In sepsis HES should be avoided due to a higher risk for acute kidney injury (AKI. Results of the usage of HES in patients without sepsis are controversial. Therefore we conducted an animal study to evaluate the impact of 6% HES 130/0.4 on kidney integrity with sepsis or under healthy conditions Sepsis was induced by standardized Colon Ascendens Stent Peritonitis (sCASP. sCASP-group as well as control group (C remained untreated for 24 h. After 18 h sCASP+HES group (sCASP+VOL and control+HES (C+VOL received 50 ml/KG balanced 6% HES (VOL 130/0.4 over 6 h. After 24 h kidney function was measured via Inulin- and PAH-Clearance in re-anesthetized rats, and serum urea, creatinine (crea, cystatin C and Neutrophil gelatinase-associated lipocalin (NGAL as well as histopathology were analysed. In vitro human proximal tubule cells (PTC were cultured +/- lipopolysaccharid (LPS and with 0.1-4.0% VOL. Cell viability was measured with XTT-, cell toxicity with LDH-test. sCASP induced severe septic AKI demonstrated divergent results regarding renal function by clearance or creatinine measure focusing on VOL. Soleley HES (C+VOL deteriorated renal function without sCASP. Histopathology revealed significantly derangements in all HES groups compared to control. In vitro LPS did not worsen the HES induced reduction of cell viability in PTC cells. For the first time, we demonstrated, that application of 50 ml/KG 6% HES 130/0.4 over 6 hours induced AKI without inflammation in vivo. Severity of sCASP induced septic AKI might be no longer susceptible to the way of volume expansion.

  15. In-vivo quantitative proteomics reveals a key contribution of post-transcriptional mechanisms to the circadian regulation of liver metabolism.

    Directory of Open Access Journals (Sweden)

    Maria S Robles

    2014-01-01

    Full Text Available Circadian clocks are endogenous oscillators that drive the rhythmic expression of a broad array of genes, orchestrating metabolism and physiology. Recent evidence indicates that post-transcriptional and post-translational mechanisms play essential roles in modulating temporal gene expression for proper circadian function, particularly for the molecular mechanism of the clock. Due to technical limitations in large-scale, quantitative protein measurements, it remains unresolved to what extent the circadian clock regulates metabolism by driving rhythms of protein abundance. Therefore, we aimed to identify global circadian oscillations of the proteome in the mouse liver by applying in vivo SILAC mouse technology in combination with state of the art mass spectrometry. Among the 3000 proteins accurately quantified across two consecutive cycles, 6% showed circadian oscillations with a defined phase of expression. Interestingly, daily rhythms of one fifth of the liver proteins were not accompanied by changes at the transcript level. The oscillations of almost half of the cycling proteome were delayed by more than six hours with respect to the corresponding, rhythmic mRNA. Strikingly we observed that the length of the time lag between mRNA and protein cycles varies across the day. Our analysis revealed a high temporal coordination in the abundance of proteins involved in the same metabolic process, such as xenobiotic detoxification. Apart from liver specific metabolic pathways, we identified many other essential cellular processes in which protein levels are under circadian control, for instance vesicle trafficking and protein folding. Our large-scale proteomic analysis reveals thus that circadian post-transcriptional and post-translational mechanisms play a key role in the temporal orchestration of liver metabolism and physiology.

  16. Three-dimensional quantitative analysis of the bone density of mandibular condyle in dentulous and edentulous jaws: an in vivo study.

    Science.gov (United States)

    Aggarwal, Himanshi; Singh, Raghuwar D; Kumar, Manoj; Singh, Ragini; Siddhartha, Ramashanker; Jurel, Sunit Kumar; Agrawal, Kaushal K; Kumar, Pradeep

    2015-01-01

    Studies have reported that masticatory function and occlusal force are low in edentulous patients, which brings about a change in the density, thickness, and alignment of bony trabeculae. However, studies that have quantitatively measured the differential cortical and medullary bone densities of the mandibular condyle in vivo remain rare. This study determined and compared the cortical and medullary bone density of the mandibular condyle in dentulous and edentulous jaws, using multidetector computed tomography (CT). Forty mandibular condyles with no clinical signs of temporomandibular disorders were investigated in 2 groups with 10 subjects (aged 50-80 yr) in each group (group I: dentulous subjects with maintained occlusion; group II: completely edentulous patients) with multidetector CT. The density of condylar cortical and medullary bone was determined by using bone density analysis algorithms available within the proprietary software. Data were analyzed statistically with the 1-way analysis of variance test (p<0.05). The mean cortical bone density of the right and left condyles of group I was 686.11±102.78 Hounsfield unit (HU) and 775.91±89.62 HU, respectively and that of group II was 531.33±289.73 HU and 648.53±294.39 HU, respectively. The mean medullary bone density of the right and left condyles was maximum in group I subjects (429.69±102.62 HU and 486.62±108.60 HU, respectively) than in group II subjects (214.89±104.37 HU and 205.36±90.91 HU, respectively) with a statistically significant decrease in the mean scores (p<0.001). Within the limitations of this study, it can be concluded that the cortical and medullary densities of the mandibular condyle are more in dentulous than the edentulous jaws.

  17. In vivo organization of the FtsZ-ring by ZapA and ZapB revealed by quantitative super-resolution microscopy.

    Science.gov (United States)

    Buss, Jackson; Coltharp, Carla; Huang, Tao; Pohlmeyer, Chris; Wang, Shih-Chin; Hatem, Christine; Xiao, Jie

    2013-09-01

    In most bacterial cells, cell division is dependent on the polymerization of the FtsZ protein to form a ring-like structure (Z-ring) at the midcell. Despite its essential role, the molecular architecture of the Z-ring remains elusive. In this work we examine the roles of two FtsZ-associated proteins, ZapA and ZapB, in the assembly dynamics and structure of the Z-ring in Escherichia coli cells. In cells deleted of zapA or zapB, we observed abnormal septa and highly dynamic FtsZ structures. While details of these FtsZ structures are difficult to discern under conventional fluorescence microscopy, single-molecule-based super-resolution imaging method Photoactivated Localization Microscopy (PALM) reveals that these FtsZ structures arise from disordered arrangements of FtsZ clusters. Quantitative analysis finds these clusters are larger and comprise more molecules than a single FtsZ protofilament, and likely represent a distinct polymeric species that is inherent to the assembly pathway of the Z-ring. Furthermore, we find these clusters are not due to the loss of ZapB-MatP interaction in ΔzapA and ΔzapB cells. Our results suggest that the main function of ZapA and ZapB in vivo may not be to promote the association of individual protofilaments but to align FtsZ clusters that consist of multiple FtsZ protofilaments. © 2013 John Wiley & Sons Ltd.

  18. Quantitative Comparison of 21 Protocols for Labeling Hippocampal Subfields and Parahippocampal Subregions in In Vivo MRI: Towards a Harmonized Segmentation Protocol

    Science.gov (United States)

    Yushkevich, Paul A.; Amaral, Robert S. C.; Augustinack, Jean C.; Bender, Andrew R.; Bernstein, Jeffrey D.; Boccardi, Marina; Bocchetta, Martina; Burggren, Alison C.; Carr, Valerie A.; Chakravarty, M. Mallar; Chetelat, Gael; Daugherty, Ana M.; Davachi, Lila; Ding, Song-Lin; Ekstrom, Arne; Geerlings, Mirjam I.; Hassan, Abdul; Huang, Yushan; Iglesias, Eugenio; La Joie, Renaud; Kerchner, Geoffrey A.; LaRocque, Karen F.; Libby, Laura A.; Malykhin, Nikolai; Mueller, Susanne G.; Olsen, Rosanna K.; Palombo, Daniela J.; Parekh, Mansi B; Pluta, John B.; Preston, Alison R.; Pruessner, Jens C.; Ranganath, Charan; Raz, Naftali; Schlichting, Margaret L.; Schoemaker, Dorothee; Singh, Sachi; Stark, Craig E. L.; Suthana, Nanthia; Tompary, Alexa; Turowski, Marta M.; Van Leemput, Koen; Wagner, Anthony D.; Wang, Lei; Winterburn, Julie L.; Wisse, Laura E.M.; Yassa, Michael A.; Zeineh, Michael M.

    2015-01-01

    OBJECTIVE An increasing number of human in vivo magnetic resonance imaging (MRI) studies have focused on examining the structure and function of the subfields of the hippocampal formation (the dentate gyrus, CA fields 1–3, and the subiculum) and subregions of the parahippocampal gyrus (entorhinal, perirhinal, and parahippocampal cortices). The ability to interpret the results of such studies and to relate them to each other would be improved if a common standard existed for labeling hippocampal subfields and parahippocampal subregions. Currently, research groups label different subsets of structures and use different rules, landmarks, and cues to define their anatomical extents. This paper characterizes, both qualitatively and quantitatively, the variability in the existing manual segmentation protocols for labeling hippocampal and parahippocampal substructures in MRI, with the goal of guiding subsequent work on developing a harmonized substructure segmentation protocol. METHOD MRI scans of a single healthy adult human subject were acquired both at 3 Tesla and 7 Tesla. Representatives from 21 research groups applied their respective manual segmentation protocols to the MRI modalities of their choice. The resulting set of 21 segmentations was analyzed in a common anatomical space to quantify similarity and identify areas of agreement. RESULTS The differences between the 21 protocols include the region within which segmentation is performed, the set of anatomical labels used, and the extents of specific anatomical labels. The greatest overall disagreement among the protocols is at the CA1/subiculum boundary, and disagreement across all structures is greatest in the anterior portion of the hippocampal formation relative to the body and tail. CONCLUSIONS The combined examination of the 21 protocols in the same dataset suggests possible strategies towards developing a harmonized subfield segmentation protocol and facilitates comparison between published studies. PMID

  19. The effects of heat on skin barrier function and in vivo dermal absorption.

    Science.gov (United States)

    Oliveira, Gabriela; Leverett, Jesse C; Emamzadeh, Mandana; Lane, Majella E

    2014-04-10

    Enhanced delivery of ingredients across the stratum corneum (SC) is of great interest for improving the efficacy of topically applied formulations. Various methods for improving dermal penetration have been reported including galvanic devices and micro-needles. From a safety perspective it is important that such approaches do not compromise SC barrier function. This study investigates the influence of topically applied heat in vivo on the dermal uptake and penetration of a model active, allantoin from gel and lotion formulations. A custom designed device was used to deliver 42°C for 30s daily to human subjects after application of two formulations containing allantoin. The results were compared with sites treated with formulations containing no active and no heat, and a control site. In addition to penetration of allantoin, the integrity of the SC was monitored using trans-epidermal water loss (TEWL) measurements. The results showed that just 30s of 42°C topically applied heat was enough to cause significantly more penetration of allantoin from the lotion formulation compared with no application of heat. TEWL data indicated that the integrity of the skin was not compromised by the treatment. However, the application of heat did not promote enhanced penetration of the active from the gel formulation. Vehicle composition is therefore an important factor when considering thermal enhancement strategies for targeting actives to the skin.

  20. Prefibrillar Tau oligomers alter the nucleic acid protective function of Tau in hippocampal neurons in vivo.

    Science.gov (United States)

    Violet, Marie; Chauderlier, Alban; Delattre, Lucie; Tardivel, Meryem; Chouala, Meliza Sendid; Sultan, Audrey; Marciniak, Elodie; Humez, Sandrine; Binder, Lester; Kayed, Rakez; Lefebvre, Bruno; Bonnefoy, Eliette; Buée, Luc; Galas, Marie-Christine

    2015-10-01

    The accumulation of DNA and RNA oxidative damage is observed in cortical and hippocampal neurons from Alzheimer's disease (AD) brains at early stages of pathology. We recently reported that Tau is a key nuclear player in the protection of neuronal nucleic acid integrity in vivo under physiological conditions and hyperthermia, a strong inducer of oxidative stress. In a mouse model of tauopathy (THY-Tau22), we demonstrate that hyperthermia selectively induces nucleic acid oxidative damage and nucleic acid strand breaks in the nucleus and cytoplasm of hippocampal neurons that display early Tau phosphorylation but no Tau fibrils. Nucleic acid-damaged neurons were exclusively immunoreactive for prefibrillar Tau oligomers. A similar association between prefibrillar Tau oligomers and nucleic acid oxidative damage was observed in AD brains. Pretreatment with Methylene Blue (MB), a Tau aggregation inhibitor and a redox cycler, reduced hyperthermia-induced Tau oligomerization as well as nucleic acid damage. This study clearly highlights the existence of an early and critical time frame for hyperthermia-induced Tau oligomerization, which most likely occurs through increased oxidative stress, and nucleic acid vulnerability during the progression of Tau pathology. These results suggest that at early stages of AD, Tau oligomerization triggers the loss of the nucleic acid protective function of monomeric Tau. This study highlights the existence of a short therapeutic window in which to prevent the formation of pathological forms of Tau and their harmful consequences on nucleic acid integrity during the progression of Tau pathology. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Exosomes function in antigen presentation during an in vivo Mycobacterium tuberculosis infection

    Science.gov (United States)

    Smith, Victoria L.; Cheng, Yong; Bryant, Barry R.; Schorey, Jeffrey S.

    2017-01-01

    Mycobacterium tuberculosis-infected macrophages and dendritic cells are limited in their ability to present antigen to CD4+ T cells suggesting that other mechanism of antigen presentation are driving the robust T cell response observed during an M. tuberculosis infection. These mechanisms could include antigens present in apoptotic bodies, necrotic debris, exosomes or even release of non-vesicular antigen from infected cells. However, there is limited data to support any of these mechanisms as important in driving T cell activation in vivo. In the present study we use Rab27a-deficient mice which show diminished trafficking of mycobacterial components to exosomes as well as M. tuberculosis strains that express recombinant proteins which traffic or fail to traffic to exosomes. We observed that exosomes released during a mouse M. tuberculosis infection contribute significantly to its T cell response. These finding imply that exosomes function to promote T cell immunity during a bacterial infection and are an important source of extracellular antigen. PMID:28262829

  2. In vivo administration of MKT-077 causes partial yet reversible impairment of mitochondrial function.

    Science.gov (United States)

    Weisberg, E L; Koya, K; Modica-Napolitano, J; Li, Y; Chen, L B

    1996-02-01

    The effects of in vivo administration of a pharmacologically toxic dose of the lipophilic cationic compound, MKT-077, were investigated in selected vital organs of the rat. MKT-077 (15 mg/kg body weight), administered by bolus i.v. injection every day for 5 days, did not detectably influence rat heart and kidney mitochondrial respiration. Although the same dosage of MKT-077 significantly decreased respiratory rates in rat liver mitochondria relative to untreated controls, complete recovery was evident within 3 days following drug withdrawal. Whereas the mitochondrial DNA of rat kidney and liver appeared to be unaffected by MKT-077 treatment, levels of heart mtDNA were noticeably less than control levels in the immediate interval following drug administration. However, this latter effect was partially reversed as early as 10 days following treatment and completely reversed within a 30-day posttreatment period. These results strongly suggest that a pharmacologically toxic dose of MKT-077 minimally affects the overall functional integrity of mitochondria in such critical, although highly vulnerable, tissues as the heart, liver, and kidney.

  3. In vivo measurement of dynamic rectus femoris function at postures representative of early swing phase.

    Science.gov (United States)

    Hernández, Antonio; Dhaher, Yasin; Thelen, Darryl G

    2008-01-01

    Forward dynamic models suggest that muscle-induced joint motions depend on dynamic coupling between body segments. As a result, biarticular muscles may exhibit non-intuitive behavior in which the induced joint motion is opposite to that assumed based on anatomy. Empirical validation of such predictions is important for models to be relied upon to characterize muscle function. In this study, we measured, in vivo, the hip and knee accelerations induced by electrical stimulation of the rectus femoris (RF) and the vastus medialis (VM) at postures representatives of the toe-off and early swing phases of the gait cycle. Seven healthy young subjects were positioned side-lying with their lower limb supported on air bearings while a 90 ms pulse train stimulated each muscle separately or simultaneously. Lower limb kinematics were measured and compared to predictions from a similarly configured dynamic model of the lower limb. We found that both RF and VM, when stimulated independently, accelerated the hip and knee into extension at these postures, consistent with model predictions. Predicted ratios of hip acceleration to knee acceleration were generally within 1 s.d. of average values. In addition, measured responses to simultaneous RF and VM stimulation were within 13% of predictions based on the assumption that joint accelerations induced by activating two muscles simultaneously can be found by adding the joint accelerations induced by activating the same muscles independently. These results provide empirical evidence of the importance of considering dynamic effects when interpreting the role of muscles in generating movement.

  4. Repetitive in vivo treatment with human recombinant interleukin-1 beta modifies beta-cell function in normal rats

    DEFF Research Database (Denmark)

    Wogensen, L D; Reimers, J; Nerup, J

    1992-01-01

    It is unknown whether interleukin-1 exerts a bimodal effect on Beta-cell function in vivo, and whether interleukin-1 has a diabetogenic action in normal animals. We therefore studied: (a) acute effects 2 h after an intraperitoneal bolus injection of 4 micrograms of recombinant human interleukin-1...

  5. Antibody-mediated targeting of the urokinase-type plasminogen activator proteolytic function neutralizes fibrinolysis in vivo

    DEFF Research Database (Denmark)

    Lund, Ida K; Jögi, Annika; Rønø, Birgitte

    2008-01-01

    Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using gene-targeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse...... models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two...

  6. Inhibitory Monoclonal Antibodies against Mouse Proteases Raised in Gene-Deficient Mice Block Proteolytic Functions in vivo

    DEFF Research Database (Denmark)

    Lund, Ida K; Rasch, Morten G; Ingvarsen, Signe

    2012-01-01

    internalization receptor uPARAP, have been developed. The inhibitory mAbs against uPA and uPAR block plasminogen activation and thereby hepatic fibrinolysis in vivo. Wound healing, another plasmin-dependent process, is delayed by an inhibitory mAb against uPA in the adult mouse. Thromboembolism can be inhibited......Identification of targets for cancer therapy requires the understanding of the in vivo roles of proteins, which can be derived from studies using gene-targeted mice. An alternative strategy is the administration of inhibitory monoclonal antibodies (mAbs), causing acute disruption of the target...... by anti-PAI-1 mAbs in vivo. In conclusion, function-blocking mAbs are well-suited for targeted therapy in mouse models of different diseases, including cancer....

  7. Treatment assessment of radiotherapy using MR functional quantitative imaging

    Institute of Scientific and Technical Information of China (English)

    Zheng; Chang; Chunhao; Wang

    2015-01-01

    Recent developments in magnetic resonance(MR) functional quantitative imaging have made it a potentially powerful tool to assess treatment response in radiation therapy. With its abilities to capture functional information on underlying tissue characteristics, MR functional quantitative imaging can be valuable in assessing treatment response and as such to optimize therapeutic outcome. Various MR quantitative imaging techniques, including diffusion weighted imaging, diffusion tensor imaging, MR spectroscopy and dynamic contrastenhanced imaging, have been investigated and found useful for assessment of radiotherapy. However, various aspects including data reproducibility, interpretation of biomarkers, image quality and data analysis impose challenges on applications of MR functional quantitative imaging in radiotherapy assessment. All of these challenging issues shall be addressed to help us understand whether MR functional quantitative imaging is truly beneficial and contributes to future development of radiotherapy. It is evident that individualized therapy is the future direction of patient care. MR functional quantitative imaging might serves as an indispensable tool towards this promising direction.

  8. Qualitative and quantitative intravaginal targeting: Key to anti-HIV-1 microbicide delivery from test tube to in vivo success

    CSIR Research Space (South Africa)

    Pillay, V

    2012-06-01

    Full Text Available employed. We hereby propose a thorough scientific qualitative and quantitative investigation of important aspects involved in HIV-1 transmission as a prerequisite for microbicide delivery. Intravaginal targeting of HIV-1 increases the chances of microbicide...

  9. The effects of Arcanobacterium pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo.

    Science.gov (United States)

    Miller, A N A; Williams, E J; Sibley, K; Herath, S; Lane, E A; Fishwick, J; Nash, D M; Rycroft, A N; Dobson, H; Bryant, C E; Sheldon, I M

    2007-10-15

    Uterine bacterial infection after parturition causes endometritis, perturbs ovarian function and leads to infertility in cattle. Although endometritis is caused by mixed infections, endometrial pathology is associated with the presence of Arcanobacterium pyogenes. The aims of the present study were to determine the effects of A. pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo. Heat-killed A. pyogenes did not affect the production of prostaglandin F2alpha (PGF) or prostaglandin E(2) (PGE) from endometrial explants, or purified populations of endometrial epithelial or stromal cells. However, the explants produced more PGF and PGE than controls when treated with a bacteria-free filtrate (BFF) cultured from A. pyogenes. Similarly, BFF stimulated PGF and PGE production by epithelial and stromal cells, respectively. So, BFF or control PBS was infused into the uterus of heifers (n=7 per group) for 8 days, starting the day after estrus. Emergence of the follicle wave, dominant follicle or corpus luteum diameter, and peripheral plasma FSH, LH, estradiol, progesterone, PGFM, or acute phase protein concentrations were unaffected by the BFF infusion. In the live animal it is likely that the intact uterine mucosa limits the exposure of the endometrial cells to the exotoxin of A. pyogenes, whereas the cells are readily exposed to the toxin in vitro.

  10. Exposure-in-vivo containing interventions to improve work functioning of workers with anxiety disorder: a systematic review

    Directory of Open Access Journals (Sweden)

    Nieuwenhuijsen Karen

    2010-10-01

    Full Text Available Abstract Background Anxiety disorders are associated with functional disability, sickness absence, and decreased productivity. Effective treatments of anxiety disorders can result in remission of symptoms. However the effects on work related outcomes are largely unknown. Exposure in vivo is potentially well fit to improve work-related outcomes. This study systematically reviews the effectiveness of exposure-in-vivo containing interventions in reducing work-related adverse outcomes in workers with anxiety disorders. Methods A systematic study search was conducted in Medline, Cinahl, Embase and Psycinfo. Two reviewers independently extracted data and from each study assessed the quality of evidence by using the GRADE approach. We performed a meta-analysis if data showed sufficient clinical homogeneity. Results Seven studies containing 11 exposure-in-vivo interventions were included. Four studies were focused on Obsessive Compulsive Disorder (OCD, two on Post Traumatic Stress Disorder (PTSD, and one on a mixed group of OCD and severe phobias. The studies were grouped according to type of anxiety disorder and subsequently according to type of comparisons. For OCD, exposure-in-vivo containing interventions can yield better work-related outcomes compared to medication (SSRIs and relaxation but not better compared to response prevention. The results on anxiety outcomes were similar. The net contribution of exposure in vivo in two OCD intervention programs is also presented as a meta-analysis and shows significant positive results on work role limitations. The calculated pooled effect size with 95% confidence interval was 0.72 (0.28, 1.15. For PTSD, exposure-in-vivo containing interventions can yield better work-related and anxiety-related outcomes compared to a waiting-list but not better compared to imaginal exposure. Conclusions Exposure in vivo as part of an anxiety treatment can reduce work-related adverse outcomes in workers with OCD and PTSD

  11. Impact of RNA editing on functions of the serotonin 2C receptor in vivo

    Directory of Open Access Journals (Sweden)

    Uade B Olaghere Da Silva

    2010-03-01

    Full Text Available Transcripts encoding 5-HT2C receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT2C-VGV receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT2C-VGV receptor (VGV/Y, we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT2C-VGV receptors. However, enhanced behavioral sensitivity to a 5-HT2C receptor agonist was also seen in mice expressing 5-HT2C-VGV receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT2C-VGV receptors had greater sensitivity to a 5-HT2C inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT2C receptor binding sites in the brains of mice solely expressing -5HT2C-VGV receptors. We conclude that 5-HT2C-VGV receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5HT2C receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT2C receptor binding sites in brain. We further caution that the pattern of 5-HT2C receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the receptor.

  12. Identifying the Functional Flexion-extension Axis of the Knee: An In-Vivo Kinematics Study.

    Directory of Open Access Journals (Sweden)

    Li Yin

    Full Text Available This study aimed to calculate the flexion-extension axis (FEA of the knee through in-vivo knee kinematics data, and then compare it with two major anatomical axes of the femoral condyles: the transepicondylar axis (TEA defined by connecting the medial sulcus and lateral prominence, and the cylinder axis (CA defined by connecting the centers of posterior condyles.The knee kinematics data of 20 healthy subjects were acquired under weight-bearing condition using bi-planar x-ray imaging and 3D-2D registration techniques. By tracking the vertical coordinate change of all points on the surface of femur during knee flexion, the FEA was determined as the line connecting the points with the least vertical shift in the medial and lateral condyles respectively. Angular deviation and distance among the TEA, CA and FEA were measured.The TEA-FEA angular deviation was significantly larger than that of the CA-FEA in 3D and transverse plane (3.45° vs. 1.98°, p < 0.001; 2.72° vs. 1.19°, p = 0.002, but not in the coronal plane (1.61° vs. 0.83°, p = 0.076. The TEA-FEA distance was significantly greater than that of the CA-FEA in the medial side (6.7 mm vs. 1.9 mm, p < 0.001, but not in the lateral side (3.2 mm vs. 2.0 mm, p = 0.16.The CA is closer to the FEA compared with the TEA; it can better serve as an anatomical surrogate for the functional knee axis.

  13. Enzymatic characterization and in vivo function of five terminal oxidases in Pseudomonas aeruginosa.

    Science.gov (United States)

    Arai, Hiroyuki; Kawakami, Takuro; Osamura, Tatsuya; Hirai, Takehiro; Sakai, Yoshiaki; Ishii, Masaharu

    2014-12-01

    The ubiquitous opportunistic pathogen Pseudomonas aeruginosa has five aerobic terminal oxidases: bo(3)-type quinol oxidase (Cyo), cyanide-insensitive oxidase (CIO), aa3-type cytochrome c oxidase (aa3), and two cbb(3)-type cytochrome c oxidases (cbb(3)-1and cbb(3)-2). These terminal oxidases are differentially regulated under various growth conditions and are thought to contribute to the survival of this microorganism in a wide variety of environmental niches. Here, we constructed multiple mutant strains of P. aeruginosa that express only one aerobic terminal oxidase to investigate the enzymatic characteristics and in vivo function of each enzyme. The Km values of Cyo, CIO, and aa3 for oxygen were similar and were 1 order of magnitude higher than those of cbb(3)-1 and cbb(3)-2, indicating that Cyo, CIO, and aa3 are low-affinity enzymes and that cbb(3)-1 and cbb(3)-2 are high-affinity enzymes. Although cbb(3)-1 and cbb(3)-2 exhibited different expression patterns in response to oxygen concentration, they had similar Km values for oxygen. Both cbb(3)-1 and cbb(3)-2 utilized cytochrome c4 as the main electron donor under normal growth conditions. The electron transport chains terminated by cbb(3)-1 and cbb(3)-2 generate a proton gradient across the cell membrane with similar efficiencies. The electron transport chain of aa3 had the highest proton translocation efficiency, whereas that of CIO had the lowest efficiency. The enzymatic properties of the terminal oxidases reported here are partially in agreement with their regulatory patterns and may explain the environmental adaptability and versatility of P. aeruginosa.

  14. Maternal separation affects dopamine transporter function in the spontaneously hypertensive rat: an in vivo electrochemical study.

    Science.gov (United States)

    Womersley, Jacqueline S; Hsieh, Jennifer H; Kellaway, Lauriston A; Gerhardt, Greg A; Russell, Vivienne A

    2011-12-01

    Attention-deficit/hyperactivity disorder (ADHD) is a developmental disorder characterised by symptoms of inattention, impulsivity and hyperactivity. The spontaneously hypertensive rat (SHR) is a well-characterised model of this disorder and has been shown to exhibit dopamine dysregulation, one of the hypothesised causes of ADHD. Since stress experienced in the early stages of life can have long-lasting effects on behaviour, it was considered that early life stress may alter development of the dopaminergic system and thereby contribute to the behavioural characteristics of SHR. It was hypothesized that maternal separation would alter dopamine regulation by the transporter (DAT) in ways that distinguish SHR from control rat strains. SHR and control Wistar-Kyoto (WKY) rats were subjected to maternal separation for 3 hours per day from postnatal day 2 to 14. Rats were tested for separation-induced anxiety-like behaviour followed by in vivo chronoamperometry to determine whether changes had occurred in striatal clearance of dopamine by DAT. The rate of disappearance of ejected dopamine was used as a measure of DAT function. Consistent with a model for ADHD, SHR were more active than WKY in the open field. SHR entered the inner zone more frequently and covered a significantly greater distance than WKY. Maternal separation increased the time that WKY spent in the closed arms and latency to enter the open arms of the elevated plus maze, consistent with other rat strains. Of note is that, maternal separation failed to produce anxiety-like behaviour in SHR. Analysis of the chronoamperometric data revealed that there was no difference in DAT function in the striatum of non-separated SHR and WKY. Maternal separation decreased the rate of dopamine clearance (k-1) in SHR striatum. Consistent with this observation, the dopamine clearance time (T100) was increased in SHR. These results suggest that the chronic mild stress of maternal separation impaired the function of striatal

  15. Maternal separation affects dopamine transporter function in the Spontaneously Hypertensive Rat: An in vivo electrochemical study

    Directory of Open Access Journals (Sweden)

    Womersley Jacqueline S

    2011-12-01

    Full Text Available Abstract Background Attention-deficit/hyperactivity disorder (ADHD is a developmental disorder characterised by symptoms of inattention, impulsivity and hyperactivity. The spontaneously hypertensive rat (SHR is a well-characterised model of this disorder and has been shown to exhibit dopamine dysregulation, one of the hypothesised causes of ADHD. Since stress experienced in the early stages of life can have long-lasting effects on behaviour, it was considered that early life stress may alter development of the dopaminergic system and thereby contribute to the behavioural characteristics of SHR. It was hypothesized that maternal separation would alter dopamine regulation by the transporter (DAT in ways that distinguish SHR from control rat strains. Methods SHR and control Wistar-Kyoto (WKY rats were subjected to maternal separation for 3 hours per day from postnatal day 2 to 14. Rats were tested for separation-induced anxiety-like behaviour followed by in vivo chronoamperometry to determine whether changes had occurred in striatal clearance of dopamine by DAT. The rate of disappearance of ejected dopamine was used as a measure of DAT function. Results Consistent with a model for ADHD, SHR were more active than WKY in the open field. SHR entered the inner zone more frequently and covered a significantly greater distance than WKY. Maternal separation increased the time that WKY spent in the closed arms and latency to enter the open arms of the elevated plus maze, consistent with other rat strains. Of note is that, maternal separation failed to produce anxiety-like behaviour in SHR. Analysis of the chronoamperometric data revealed that there was no difference in DAT function in the striatum of non-separated SHR and WKY. Maternal separation decreased the rate of dopamine clearance (k-1 in SHR striatum. Consistent with this observation, the dopamine clearance time (T100 was increased in SHR. These results suggest that the chronic mild stress of

  16. Self-Assembled Tb(3+) Complex Probe for Quantitative Analysis of ATP during Its Enzymatic Hydrolysis via Time-Resolved Luminescence in Vitro and in Vivo.

    Science.gov (United States)

    Jung, Sung Ho; Kim, Ka Young; Lee, Ji Ha; Moon, Cheol Joo; Han, Noh Soo; Park, Su-Jin; Kang, Dongmin; Song, Jae Kyu; Lee, Shim Sung; Choi, Myong Yong; Jaworski, Justyn; Jung, Jong Hwa

    2017-01-11

    To more accurately assess the pathways of biological systems, a probe is needed that may respond selectively to adenosine triphosphate (ATP) for both in vitro and in vivo detection modes. We have developed a luminescence probe that can provide real-time information on the extent of ATP, ADP, and AMP by virtue of the luminescence and luminescence lifetime observed from a supramolecular polymer based on a C3 symmetrical terpyridine complex with Tb(3+) (S1-Tb). The probe shows remarkable selective luminescence enhancement in the presence of ATP compared to other phosphate-displaying nucleotides including adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), thymidine triphosphate (TTP), H2PO4(-) (Pi), and pyrophosphate (PPi). In addition, the time-resolved luminescence lifetime and luminescence spectrum of S1-Tb could facilitate the quantitative measurement of the exact amount of ATP and similarly ADP and AMP within living cells. The time-resolved luminescence lifetime of S1-Tb could also be used to quantitatively monitor the amount of ATP, ADP, and AMP in vitro following the enzymatic hydrolysis of ATP. The long luminescence lifetime, which was observed into the millisecond range, makes this S1-Tb-based probe particularly attractive for monitoring biological ATP levels in vivo, because any short lifetime background fluorescence arising from the complex molecular environment may be easily eliminated.

  17. Construction of Lyapunov functions for some models of infectious diseases in vivo: from simple models to complex models.

    Science.gov (United States)

    Kajiwara, Tsuyoshi; Sasaki, Toru; Takeuchi, Yasuhiro

    2015-02-01

    We present a constructive method for Lyapunov functions for ordinary differential equation models of infectious diseases in vivo. We consider models derived from the Nowak-Bangham models. We construct Lyapunov functions for complex models using those of simpler models. Especially, we construct Lyapunov functions for models with an immune variable from those for models without an immune variable, a Lyapunov functions of a model with absorption effect from that for a model without absorption effect. We make the construction clear for Lyapunov functions proposed previously, and present new results with our method.

  18. In vivo subsurface morphological and functional cellular and subcellular imaging of the gastrointestinal tract with confocal mini-microscopy

    Institute of Scientific and Technical Information of China (English)

    Martin Goetz; Beena Memadathil; Stefan Biesterfeld; Constantin Schneider; Sebastian Gregor; Peter R Galle; Markus F Neurath; Ralf Kiesslich

    2007-01-01

    AIM: To evaluate a newly developed hand-held confocal probe for in vivo microscopic imaging of the complete gastrointestinal tract in rodents.METHODS: A novel rigid confocal probe (diameter 7 mm) was designed with optical features similar to the flexible endomicroscopy system for use in humans using a 488 nm single line laser for fluorophore excitation.Light emission was detected at 505 to 750 nm. The field of view was 475 μm × 475 μm. Optical slice thickness was 7 μm with a lateral resolution of 0.7 μm. Subsurface serial images at different depths (surface to 250 μm)were generated in real time at 1024 × 1024 pixels (0.8 frames/s) by placing the probe onto the tissue in gentle,stable contact. Tissue specimens were sampled for histopathological correlation.RESULTS: The esophagus, stomach, small and large intestine and meso, liver, pancreas and gall bladder were visualised in vivo at high resolution in n = 48 mice.Real time microscopic imaging with the confocal minimicroscopy probe was easy to achieve. The different staining protocols (fluorescein, acriflavine, FITC-labelled dextran and L. esculentum lectin) each highlighted specific aspects of the tissue, and in vivo imaging correlated excellently with conventional histology. In vivo blood flow monitoring added a functional quality to morphologic imaging.CONCLUSION: Confocal microscopy is feasible in vivo allowing the visualisation of the complete GI tract at high resolution even of subsurface tissue structures.The new confocal probe design evaluated in this study is compatible with laparoscopy and significantly expands the field of possible applications to intra-abdominal organs. It allows immediate testing of new in vivo staining and application options and therefore permits rapid transfer from animal studies to clinical use in patients.

  19. Functional analysis of Pro-inflammatory properties within the cerebrospinal fluid after subarachnoid hemorrhage in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Schneider Ulf C

    2012-02-01

    Full Text Available Abstract Background To functionally characterize pro-inflammatory and vasoconstrictive properties of cerebrospinal fluid after aneurysmal subarachnoid hemorrhage (SAH in vivo and in vitro. Methods The cerebrospinal fluid (CSF of 10 patients suffering from SAH was applied to the transparent skinfold chamber model in male NMRI mice which allows for in vivo analysis of the microcirculatory response to a superfusat. Microvascular diameter changes were quantified and the numbers of rolling and sticking leukocytes were documented using intravital multifluorescence imaging techniques. Furthermore, the pro-inflammatory properties of CSF were assessed in vitro using a monocyte transendothelial migration assay. Results CSF superfusion started to induce significant vasoconstriction on days 4 and 6 after SAH. In parallel, CSF superfusion induced a microvascular leukocyte recruitment, with a significant number of leukocytes rolling (day 6 and sticking (days 2-4 to the endothelium. CSF of patients presenting with cerebral edema induced breakdown of blood vessel integrity in our assay as evidenced by fluorescent marker extravasation. In accordance with leukocyte activation in vivo, significantly higher in vitro monocyte migration rates were found after SAH. Conclusion We functionally characterized inflammatory and vasoactive properties of patients' CSF after SAH in vivo and in vitro. This pro-inflammatory milieu in the subarachnoid space might play a pivotal role in the pathophysiology of early and delayed brain injury as well as vasospasm development following SAH.

  20. Microtubule depolymerization normalizes in vivo myocardial contractile function in dogs with pressure-overload left ventricular hypertrophy

    Science.gov (United States)

    Koide, M.; Hamawaki, M.; Narishige, T.; Sato, H.; Nemoto, S.; DeFreyte, G.; Zile, M. R.; Cooper G, I. V.; Carabello, B. A.

    2000-01-01

    BACKGROUND: Because initially compensatory myocardial hypertrophy in response to pressure overloading may eventually decompensate to myocardial failure, mechanisms responsible for this transition have long been sought. One such mechanism established in vitro is densification of the cellular microtubule network, which imposes a viscous load that inhibits cardiocyte contraction. METHODS AND RESULTS: In the present study, we extended this in vitro finding to the in vivo level and tested the hypothesis that this cytoskeletal abnormality is important in the in vivo contractile dysfunction that occurs in experimental aortic stenosis in the adult dog. In 8 dogs in which gradual stenosis of the ascending aorta had caused severe left ventricular (LV) pressure overloading (gradient, 152+/-16 mm Hg) with contractile dysfunction, LV function was measured at baseline and 1 hour after the intravenous administration of colchicine. Cardiocytes obtained by biopsy before and after in vivo colchicine administration were examined in tandem. Microtubule depolymerization restored LV contractile function both in vivo and in vitro. CONCLUSIONS: These and additional corroborative data show that increased cardiocyte microtubule network density is an important mechanism for the ventricular contractile dysfunction that develops in large mammals with adult-onset pressure-overload-induced cardiac hypertrophy.

  1. In vivo visualization of expression and function of miR221 in papillary thyroid carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyun Joo; Kim, Soon Hag; Jeong, Jae Min; Lee, Dong Soo; Chung, June Key [Seoul National Univ. College of Medicine, Seoul (Korea, Republic of)

    2007-07-01

    MicroRNA 221 has been known to be one of up-regulated miRNAs in papillary thyroid carcinoma. To evaluate the expression and function of miR221, we measured the quantities of primary or mature miR221 in normal thyroid or papillary cancer cells and examined the gene expression of Gaussian luciferase (Glue) regulated by miR221. Total RNA and small RNA were isolated from normal thyroid cells (HT-ori3) and papillary thyroid cancer cells (NPA, TPC-1). The quantities of primary miR221 or mature miR221 in cells were measured by qRT-PCR. We constructed a CMV/Gluc-3xPT{sub m}iR221 including 3 times repeated perfect target sequences of miR221 in the 3'UTR of Gluc. CMV/Gluc-3xPT{sub m}iR221 was transfected into HT-ori3, NPA, and TPC-1, CMV/Gluc was used as a control. Also, precursor miR221 or anti-miR221 were co-transfected with CMV/Gluc-3xPT{sub m}iR221 into each cells and compared the Gluc activities by luciferase assay and in vivo bioluminescence image. The quantities of primary miR221 of NPA or TPC-1 were 2.24 or 1.5 times more than that of HT-ori3, and quantities of mature miR221 in NPA or TPC-1 were 17 or 7 times more than that of HT-ori3, respectively. Gluc activities in NPA or TPC-1 transfected with CMV/Gluc-3xPT{sub m}iR221 were repressed 2 times or more than those of CMV/Gluc, respectively. Also, Gluc activities in NPA or TPC-1 co-transfected with pre-miR221 and CMV/Gluc-3xPT{sub m}iR221 were repressed 5 times or more down-regulated than those of CMV/Gluc, respectively. The other hand, Gluc activities in NPA or TPC-1 co-transfected with anti-miR221 and CMV/Gluc-3xPT{sub m}iR221 were retrieved as those of CMV/Gluc. In vivo bioluminescence images also showed that Gluc activities were repressed by pre.miR221 and retrieved by anti-miR221. These results suggest that CMV/Gluc-3xPT{sub m}iR221 system may be a useful tool for monitoring the quantities of miR221 and gene regulation by miR221 in a living animal.

  2. Characterization of in vivo Dlg1 deletion on T cell development and function.

    Directory of Open Access Journals (Sweden)

    Lisa A Humphries

    Full Text Available BACKGROUND: The polarized reorganization of the T cell membrane and intracellular signaling molecules in response to T cell receptor (TCR engagement has been implicated in the modulation of T cell development and effector responses. In siRNA-based studies Dlg1, a MAGUK scaffold protein and member of the Scribble polarity complex, has been shown to play a role in T cell polarity and TCR signal specificity, however the role of Dlg1 in T cell development and function in vivo remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the combined data from three independently-derived dlg1-knockout mouse models; two germline deficient knockouts and one conditional knockout. While defects were not observed in T cell development, TCR-induced early phospho-signaling, actin-mediated events, or proliferation in any of the models, the acute knockdown of Dlg1 in Jurkat T cells diminished accumulation of actin at the IS. Further, while Th1-type cytokine production appeared unaffected in T cells derived from mice with a dlg1 germline-deficiency, altered production of TCR-dependent Th1 and Th2-type cytokines was observed in T cells derived from mice with a conditional loss of dlg1 expression and T cells with acute Dlg1 suppression, suggesting a differential requirement for Dlg1 activity in signaling events leading to Th1 versus Th2 cytokine induction. The observed inconsistencies between these and other knockout models and siRNA strategies suggest that 1 compensatory upregulation of alternate gene(s may be masking a role for dlg1 in controlling TCR-mediated events in dlg1 deficient mice and 2 the developmental stage during which dlg1 ablation begins may control the degree to which compensatory events occur. CONCLUSIONS/SIGNIFICANCE: These findings provide a potential explanation for the discrepancies observed in various studies using different dlg1-deficient T cell models and underscore the importance of acute dlg1 ablation to avoid the upregulation of

  3. Characterization of In Vivo Dlg1 Deletion on T Cell Development and Function

    Science.gov (United States)

    Tomassian, Tamar; McMahon, Kerrie-Ann; Humbert, Patrick O.; Silva, Oscar; Round, June L.; Takamiya, Kogo; Huganir, Richard L.

    2012-01-01

    Background The polarized reorganization of the T cell membrane and intracellular signaling molecules in response to T cell receptor (TCR) engagement has been implicated in the modulation of T cell development and effector responses. In siRNA-based studies Dlg1, a MAGUK scaffold protein and member of the Scribble polarity complex, has been shown to play a role in T cell polarity and TCR signal specificity, however the role of Dlg1 in T cell development and function in vivo remains unclear. Methodology/Principal Findings Here we present the combined data from three independently-derived dlg1-knockout mouse models; two germline deficient knockouts and one conditional knockout. While defects were not observed in T cell development, TCR-induced early phospho-signaling, actin-mediated events, or proliferation in any of the models, the acute knockdown of Dlg1 in Jurkat T cells diminished accumulation of actin at the IS. Further, while Th1-type cytokine production appeared unaffected in T cells derived from mice with a dlg1germline-deficiency, altered production of TCR-dependent Th1 and Th2-type cytokines was observed in T cells derived from mice with a conditional loss of dlg1 expression and T cells with acute Dlg1 suppression, suggesting a differential requirement for Dlg1 activity in signaling events leading to Th1 versus Th2 cytokine induction. The observed inconsistencies between these and other knockout models and siRNA strategies suggest that 1) compensatory upregulation of alternate gene(s) may be masking a role for dlg1 in controlling TCR-mediated events in dlg1 deficient mice and 2) the developmental stage during which dlg1 ablation begins may control the degree to which compensatory events occur. Conclusions/Significance These findings provide a potential explanation for the discrepancies observed in various studies using different dlg1-deficient T cell models and underscore the importance of acute dlg1 ablation to avoid the upregulation of compensatory

  4. Development of optical neuroimaging to detect drug-induced brain functional changes in vivo

    Science.gov (United States)

    Du, Congwu; Pan, Yingtian

    2014-03-01

    Deficits in prefrontal function play a crucial role in compulsive cocaine use, which is a hallmark of addiction. Dysfunction of the prefrontal cortex might result from effects of cocaine on neurons as well as from disruption of cerebral blood vessels. However, the mechanisms underlying cocaine's neurotoxic effects are not fully understood, partially due to technical limitations of current imaging techniques (e.g., PET, fMRI) to differentiate vascular from neuronal effects at sufficiently high temporal and spatial resolutions. We have recently developed a multimodal imaging platform which can simultaneously characterize the changes in cerebrovascular hemodynamics, hemoglobin oxygenation and intracellular calcium fluorescence for monitoring the effects of cocaine on the brain. Such a multimodality imaging technique (OFI) provides several uniquely important merits, including: 1) a large field-of-view, 2) high spatiotemporal resolutions, 3) quantitative 3D imaging of the cerebral blood flow (CBF) networks, 4) label-free imaging of hemodynamic changes, 5) separation of vascular compartments (e.g., arterial and venous vessels) and monitoring of cortical brain metabolic changes, 6) discrimination of cellular (neuronal) from vascular responses. These imaging features have been further advanced in combination with microprobes to form micro-OFI that allows quantification of drug effects on subcortical brain. In addition, our ultrahigh-resolution ODT (μODT) enables 3D microangiography and quantitative imaging of capillary CBF networks. These optical strategies have been used to investigate the effects of cocaine on brain physiology to facilitate the studies of brain functional changes induced by addictive substance to provide new insights into neurobiological effects of the drug on the brain.

  5. Radiopharmaceuticals: nanoparticles like multi-functional systems for the obtaining in vivo of molecular images; Radiofarmacos: nanoparticulas como sistemas multifuncionales para la obtencion in vivo de imagenes moleculares

    Energy Technology Data Exchange (ETDEWEB)

    Ferro F, G.; Ramirez de la Cruz, F. M.; Ocampo G, B. E.; Morales A, E.; Santos C, C. L.; Mendoza S, A. N., E-mail: guillermina.ferro@inin.gob.m [ININ, Departamento de Materiales Radiactivos, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2010-07-01

    The techniques of obtaining direct or indirect molecular images detect and register the space-temporary distribution of molecular or cellular processes for biochemical, biological, diagnostic and therapeutic applications. The advanced techniques of image like the nuclear magnetic resonance, the single photon emission computed tomography, the positron emission tomography and the images of optic fluorescence have been used successfully to detect these processes. On the other hand, the utility of the nanoparticles for any application is dependent of the physicochemical properties that present, being possible to modify their surface when making them react with different biomolecules what allows the formation of conjugates with specific molecular recognition. The joint of various protein molecules, peptides or oligonucleotides to the surface of a nanoparticle produce a multi-functional system able to increase the multivalent joints from the nanoparticles-biomolecules to their receivers for the obtaining of molecular images in vivo. The peptides stimulate, regulate or inhibit numerous functions of the life, acting mainly as information transmitters and activity coordinators of several tissues in the organism. The receivers of regulator peptides are over represented in numerous types of cancer cells and they are protein structures. These receivers have been used as white molecular of marked peptides, to locate primary malignant tumors and their metastasis, using the diagnostic techniques of molecular image mentioned above, which consist basically on the radio peptides use and conjugated peptides to fluoro chromes, to metallic nanoparticles and nano crystals. A summary of the work is presented carried out by the personnel of the Radio-active Materials and Chemistry Departments of the Instituto Nacional de Investigaciones Nucleares in this field. (Author)

  6. Molecular imaging of melanin distribution in vivo and quantitative differential diagnosis of human pigmented lesions using label-free harmonic generation biopsy (Conference Presentation)

    Science.gov (United States)

    Sun, Chi-Kuang; Wei, Ming-Liang; Su, Yu-Hsiang; Weng, Wei-Hung; Liao, Yi-Hua

    2017-02-01

    Harmonic generation microscopy is a noninvasive repetitive imaging technique that provides real-time 3D microscopic images of human skin with a sub-femtoliter resolution and high penetration down to the reticular dermis. In this talk, we show that with a strong resonance effect, the third-harmonic-generation (THG) modality provides enhanced contrast on melanin and allows not only differential diagnosis of various pigmented skin lesions but also quantitative imaging for longterm tracking. This unique capability makes THG microscopy the only label-free technique capable of identifying the active melanocytes in human skin and to image their different dendriticity patterns. In this talk, we will review our recent efforts to in vivo image melanin distribution and quantitatively diagnose pigmented skin lesions using label-free harmonic generation biopsy. This talk will first cover the spectroscopic study on the melanin enhanced THG effect in human cells and the calibration strategy inside human skin for quantitative imaging. We will then review our recent clinical trials including: differential diagnosis capability study on pigmented skin tumors; as well as quantitative virtual biopsy study on pre- and post- treatment evaluation on melasma and solar lentigo. Our study indicates the unmatched capability of harmonic generation microscopy to perform virtual biopsy for noninvasive histopathological diagnosis of various pigmented skin tumors, as well as its unsurpassed capability to noninvasively reveal the pathological origin of different hyperpigmentary diseases on human face as well as to monitor the efficacy of laser depigmentation treatments. This work is sponsored by National Health Research Institutes.

  7. Exchange of regions of the carboxypeptidase Y propeptide. Sequence specificity and function in folding in vivo

    DEFF Research Database (Denmark)

    Ramos, C; Winther, Jakob R.

    1996-01-01

    The propeptide of carboxypeptidase Y from Saccharomyces cerevisiae is important for folding of the enzyme. Previous work [Ramos, C., Winther, J.R. & Kielland-Brandt, M. C. (1994) J. Biol. Chem. 269, 7006-7012] suggested that the sequences essential for in vivo folding were situated in the COOH...

  8. Functionalized near-infrared quantum dots for in vivo tumor vasculature imaging

    Energy Technology Data Exchange (ETDEWEB)

    Hu Rui; Yong, Ken-Tye; Roy, Indrajit; Ding Hong; Law, Wing-Cheung; Cai Hongxing; Vathy, Lisa A; Bergey, Earl J; Prasad, Paras N [Institute for Lasers, Photonics and Biophotonics (ILPB), University at Buffalo, State University of New York, Buffalo, NY 14260-4200 (United States); Zhang Xihe, E-mail: kyong2@buffalo.edu, E-mail: pnprasad@buffalo.edu [ChangChun University of Science and Technology (CUST), ChangChun, Jilin 130022 (China)

    2010-04-09

    In this paper, we report the use of near-infrared (NIR)-emitting alloyed quantum dots (QDs) as efficient optical probes for high contrast in vivo imaging of tumors. Alloyed CdTe{sub 1-x}Se{sub x}/CdS QDs were prepared in the non-aqueous phase using the hot colloidal synthesis approach. Water dispersion of the QDs were accomplished by their encapsulation within polyethyleneglycol (PEG)-grafted phospholipid micelles. For tumor-specific delivery in vivo, the micelle-encapsulated QDs were conjugated with the cyclic arginine-glycine-aspartic acid (cRGD) peptide, which targets the {alpha}{sub v{beta}3} integrins overexpressed in the angiogenic tumor vasculatures. Using in vivo NIR optical imaging of mice bearing pancreatic cancer xenografts, implanted both subcutaneously and orthotopically, we have demonstrated that systemically delivered cRGD-conjugated QDs, but not the unconjugated ones, can efficiently target and label the tumors with high signal-to-noise ratio. Histopathological analysis of major organs of the treated mice showed no evidence of systemic toxicity associated with these QDs. These experiments suggest that cRGD-conjugated NIR QDs can serve as safe and efficient probes for optical bioimaging of tumors in vivo. Furthermore, by co-encapsulating these QDs and anticancer drugs within these micelles, we have demonstrated a promising theranostic, nanosized platform for both cancer imaging and therapy.

  9. Integration analysis of quantitative proteomics and transcriptomics data identifies potential targets of frizzled-8 protein-related antiproliferative factor in vivo.

    Science.gov (United States)

    Yang, Wei; Kim, Yongsoo; Kim, Taek-Kyun; Keay, Susan K; Kim, Kwang Pyo; Steen, Hanno; Freeman, Michael R; Hwang, Daehee; Kim, Jayoung

    2012-12-01

    What's known on the subject? and What does the study add? Interstitial cystitis (IC) is a prevalent and debilitating pelvic disorder generally accompanied by chronic pain combined with chronic urinating problems. Over one million Americans are affected, especially middle-aged women. However, its aetiology or mechanism remains unclear. No efficient drug has been provided to patients. Several urinary biomarker candidates have been identified for IC; among the most promising is antiproliferative factor (APF), whose biological activity is detectable in urine specimens from >94% of patients with both ulcerative and non-ulcerative IC. The present study identified several important mediators of the effect of APF on bladder cell physiology, suggesting several candidate drug targets against IC. In an attempt to identify potential proteins and genes regulated by APF in vivo, and to possibly expand the APF-regulated network identified by stable isotope labelling by amino acids in cell culture (SILAC), we performed an integration analysis of our own SILAC data and the microarray data of Gamper et al. (2009) BMC Genomics 10: 199. Notably, two of the proteins (i.e. MAPKSP1 and GSPT1) that are down-regulated by APF are involved in the activation of mTORC1, suggesting that the mammalian target of rapamycin (mTOR) pathway is potentially a critical pathway regulated by APF in vivo. Several components of the mTOR pathway are currently being studied as potential therapeutic targets in other diseases. Our analysis suggests that this pathway might also be relevant in the design of diagnostic tools and medications targeting IC. • To enhance our understanding of the interstitial cystitis urine biomarker antiproliferative factor (APF), as well as interstitial cystitis biology more generally at the systems level, we reanalyzed recently published large-scale quantitative proteomics and in vivo transcriptomics data sets using an integration analysis tool that we have developed. • To

  10. Renaissance of morphological studies: the examination of functional structures in living animal organs using the in vivo cryotechnique.

    Science.gov (United States)

    Ohno, Shinichi; Saitoh, Yurika; Ohno, Nobuhiko; Terada, Nobuo

    2017-01-01

    Medical and biological scientists wish to understand the in vivo structures of the cells and tissues that make up living animal organs, as well as the locations of their molecular components. Recently, the live imaging of animal cells and tissues with fluorescence-labeled proteins produced via gene manipulation has become increasingly common. Therefore, it is important to ensure that findings derived from histological or immunohistochemical tissue sections of living animal organs are compatible with those obtained from live images of the same organs, which can be assessed using recently developed digital imaging techniques. Over the past two decades, we have performed immunohistochemical and morphological studies of the cells and tissues in living animal organs using a novel in vivo cryotechnique. The use of a specially designed liquid cryogen system with or without a cryoknife during this cryotechnique solved the technical problems that inevitably arise during the conventional preparation methods employed prior to light or electron microscopic examinations. Our in vivo cryotechnique has been found to be extremely useful for arresting transient physiological processes in cells and tissues and for maintaining their functional components-such as rapidly changing signaling molecules, membrane channels, or receptors-in situ. The purpose of the present review is to describe the basic mechanism underlying cryotechniques and the significance of our in vivo cryotechnique. In addition, it describes various morphological or immunohistochemical findings, observations made using quantum dots, and a Raman cryomicroscopy-based method for assessing oxygen saturation in the erythrocytes flowing through intestinal tissues.

  11. Defective mitochondrial function in vivo in skeletal muscle in adults with Down's syndrome: a 31P-MRS study.

    Directory of Open Access Journals (Sweden)

    Alexander C Phillips

    Full Text Available Down's syndrome (DS is a developmental disorder associated with intellectual disability (ID. We have previously shown that people with DS engage in very low levels of exercise compared to people with ID not due to DS. Many aspects of the DS phenotype, such as dementia, low activity levels and poor muscle tone, are shared with disorders of mitochondrial origin, and mitochondrial dysfunction has been demonstrated in cultured DS tissue. We undertook a phosphorus magnetic resonance spectroscopy ((31P-MRS study in the quadriceps muscle of 14 people with DS and 11 non-DS ID controls to investigate the post-exercise resynthesis kinetics of phosphocreatine (PCr, which relies on mitochondrial respiratory function and yields a measure of muscle mitochondrial function in vivo. We found that the PCr recovery rate constant was significantly decreased in adults with DS compared to non-DS ID controls (1.7 ± 0.1 min(-1 vs 2.1 ± 0.1 min(-1 respectively who were matched for physical activity levels, indicating that muscle mitochondrial function in vivo is impaired in DS. This is the first study to investigate mitochondrial function in vivo in DS using (31P-MRS. Our study is consistent with previous in vitro studies, supporting a theory of a global mitochondrial defect in DS.

  12. Defective mitochondrial function in vivo in skeletal muscle in adults with Down's syndrome: a 31P-MRS study.

    Science.gov (United States)

    Phillips, Alexander C; Sleigh, Alison; McAllister, Catherine J; Brage, Soren; Carpenter, T Adrian; Kemp, Graham J; Holland, Anthony J

    2013-01-01

    Down's syndrome (DS) is a developmental disorder associated with intellectual disability (ID). We have previously shown that people with DS engage in very low levels of exercise compared to people with ID not due to DS. Many aspects of the DS phenotype, such as dementia, low activity levels and poor muscle tone, are shared with disorders of mitochondrial origin, and mitochondrial dysfunction has been demonstrated in cultured DS tissue. We undertook a phosphorus magnetic resonance spectroscopy ((31)P-MRS) study in the quadriceps muscle of 14 people with DS and 11 non-DS ID controls to investigate the post-exercise resynthesis kinetics of phosphocreatine (PCr), which relies on mitochondrial respiratory function and yields a measure of muscle mitochondrial function in vivo. We found that the PCr recovery rate constant was significantly decreased in adults with DS compared to non-DS ID controls (1.7 ± 0.1 min(-1) vs 2.1 ± 0.1 min(-1) respectively) who were matched for physical activity levels, indicating that muscle mitochondrial function in vivo is impaired in DS. This is the first study to investigate mitochondrial function in vivo in DS using (31)P-MRS. Our study is consistent with previous in vitro studies, supporting a theory of a global mitochondrial defect in DS.

  13. REVIEW ARTICLE: In vivo magnetic resonance imaging: insights into structure and function of the central nervous system

    Science.gov (United States)

    Natt, Oliver; Frahm, Jens

    2005-04-01

    Spatially resolved nuclear magnetic resonance (NMR) techniques provide structural, metabolic and functional insights into the central nervous system and allow for repetitive in vivo studies of both humans and animals. Complementing its prominent role in diagnostic imaging, magnetic resonance imaging (MRI) has evolved into an indispensable research tool in system-oriented neurobiology where contributions to functional genomics and translational medicine bridge the gap from molecular biology to animal models and clinical applications. This review presents an overview on some of the most relevant advances in MRI. An introduction covering the basic principles is followed by a discussion of technological improvements in instrumentation and imaging sequences including recent developments in parallel acquisition techniques. Because MRI is noninvasive in contrast to most other imaging modalities, examples focus on in vivo studies of the central nervous system in a variety of species ranging from humans to mice and insects.

  14. In vivo evaluation of biomechanical properties in the patellofemoral joint after matrix-associated autologous chondrocyte transplantation by means of quantitative T2 MRI.

    Science.gov (United States)

    Pachowsky, M L; Trattnig, S; Wondrasch, B; Apprich, S; Marlovits, S; Mauerer, A; Welsch, Goetz H; Blanke, M

    2014-06-01

    To determine in vivo biomechanical properties of articular cartilage and cartilage repair tissue of the patella, using biochemical MRI by means of quantitative T2 mapping. Twenty MR scans were achieved at 3T MRI, using a new 8-channel multi-function coil allowing controlled bending of the knee. Multi-echo spin-echo T2 mapping was prepared in healthy volunteers and in age- and sex-matched patients after matrix-associated autologous chondrocyte transplantation (MACT) of the patella. MRI was performed at 0° and 45° of flexion of the knee after 0 min and after 1 h. A semi-automatic region-of-interest analysis was performed for the whole patella cartilage. To allow stratification with regard to the anatomical (collagen) structure, further subregional analysis was carried out (deep-middle-superficial cartilage layer). Statistical analysis of variance was performed. During 0° flexion (decompression), full-thickness T2 values showed no significant difference between volunteers (43 ms) and patients (41 ms). Stratification was more pronounced for healthy cartilage compared to cartilage repair tissue. During 45° flexion (compression), full-thickness T2 values within volunteers were significantly increased (54 ms) compared to patients (44 ms) (p < 0.001). Again, stratification was more pronounced in volunteers compared to patients. The volunteer group showed no significant increase in T2 values measured in straight position and in bended position. There was no significant difference between the 0- and the 60-min MRI examination. T2 values in the patient group increased between the 0- and the 60-min examination. However, the increase was only significant in the superior cartilage layer of the straight position (p = 0.021). During compression (at 45° flexion), healthy patellar cartilage showed a significant increase in T2-values, indicating adaptations of water content and collagen fibril orientation to mechanical load. This could not be observed within the patella

  15. Quantitative comparison of PET and Bremsstrahlung SPECT for imaging the in vivo yttrium-90 microsphere distribution after liver radioembolization.

    Directory of Open Access Journals (Sweden)

    Mattijs Elschot

    Full Text Available BACKGROUND: After yttrium-90 ((90Y microsphere radioembolization (RE, evaluation of extrahepatic activity and liver dosimetry is typically performed on (90Y Bremsstrahlung SPECT images. Since these images demonstrate a low quantitative accuracy, (90Y PET has been suggested as an alternative. The aim of this study is to quantitatively compare SPECT and state-of-the-art PET on the ability to detect small accumulations of (90Y and on the accuracy of liver dosimetry. METHODOLOGY/PRINCIPAL FINDINGS: SPECT/CT and PET/CT phantom data were acquired using several acquisition and reconstruction protocols, including resolution recovery and Time-Of-Flight (TOF PET. Image contrast and noise were compared using a torso-shaped phantom containing six hot spheres of various sizes. The ability to detect extra- and intrahepatic accumulations of activity was tested by quantitative evaluation of the visibility and unique detectability of the phantom hot spheres. Image-based dose estimates of the phantom were compared to the true dose. For clinical illustration, the SPECT and PET-based estimated liver dose distributions of five RE patients were compared. At equal noise level, PET showed higher contrast recovery coefficients than SPECT. The highest contrast recovery coefficients were obtained with TOF PET reconstruction including resolution recovery. All six spheres were consistently visible on SPECT and PET images, but PET was able to uniquely detect smaller spheres than SPECT. TOF PET-based estimates of the dose in the phantom spheres were more accurate than SPECT-based dose estimates, with underestimations ranging from 45% (10-mm sphere to 11% (37-mm sphere for PET, and 75% to 58% for SPECT, respectively. The differences between TOF PET and SPECT dose-estimates were supported by the patient data. CONCLUSIONS/SIGNIFICANCE: In this study we quantitatively demonstrated that the image quality of state-of-the-art PET is superior over Bremsstrahlung SPECT for the

  16. Quantitative Liver Function Tests: A Realizable Goal?

    OpenAIRE

    Morgan, Denis J; Susan L Elliott; Hany Ghabrial; Smallwood, Richard A

    1991-01-01

    A variety of tests has been used to assess liver function and predict hepatic functional reserve in patients with liver disease. These tests comprise clinical assessment, simple biochemical measurements and so-called ‘quantitative’ tests of liver function, ie, elimination rate measurements of exogenous markers such as drugs and other compounds. So far no single test or group of tests has proved to be a sufficiently sensitive and accurate measure of overall hepatic function across the whole sp...

  17. Biodistribution of amino-functionalized diamond nanoparticles. In vivo studies based on 18F radionuclide emission.

    Science.gov (United States)

    Rojas, Santiago; Gispert, Juan D; Martín, Roberto; Abad, Sergio; Menchón, Cristina; Pareto, Deborah; Víctor, Víctor M; Alvaro, Mercedes; García, Hermenegildo; Herance, J Raúl

    2011-07-26

    Nanoparticles have been proposed for several biomedical applications; however, in vivo biodistribution studies to confirm their potential are scarce. Nanodiamonds are carbon nanoparticles that have been recently proposed as a promising biomaterial. In this study, we labeled nanodiamonds with (18)F to study their in vivo biodistribution by positron emission tomography. Moreover, the impact on the biodistribution of their kinetic particle size and of the surfactant agents has been evaluated. Radiolabeled diamond nanoparticles accumulated mainly in the lung, spleen, and liver and were excreted into the urinary tract. The addition of surfactant agents did not lead to significant changes in this pattern, with the exception of a slight reduction in the urinary excretion rate. On the other hand, after filtration of the radiolabeled diamond nanoparticles to remove those with a larger kinetic size, the uptake in the lung and spleen was completely inhibited and significantly reduced in the liver.

  18. Integration of Microfractionation, qNMR and zebrafish screening for the in vivo bioassay-guided isolation and quantitative bioactivity analysis of natural products.

    Science.gov (United States)

    Bohni, Nadine; Cordero-Maldonado, María Lorena; Maes, Jan; Siverio-Mota, Dany; Marcourt, Laurence; Munck, Sebastian; Kamuhabwa, Appolinary R; Moshi, Mainen J; Esguerra, Camila V; de Witte, Peter A M; Crawford, Alexander D; Wolfender, Jean-Luc

    2013-01-01

    Natural products (NPs) are an attractive source of chemical diversity for small-molecule drug discovery. Several challenges nevertheless persist with respect to NP discovery, including the time and effort required for bioassay-guided isolation of bioactive NPs, and the limited biomedical relevance to date of in vitro bioassays used in this context. With regard to bioassays, zebrafish have recently emerged as an effective model system for chemical biology, allowing in vivo high-content screens that are compatible with microgram amounts of compound. For the deconvolution of the complex extracts into their individual constituents, recent progress has been achieved on several fronts as analytical techniques now enable the rapid microfractionation of extracts, and microflow NMR methods have developed to the point of allowing the identification of microgram amounts of NPs. Here we combine advanced analytical methods with high-content screening in zebrafish to create an integrated platform for microgram-scale, in vivo NP discovery. We use this platform for the bioassay-guided fractionation of an East African medicinal plant, Rhynchosia viscosa, resulting in the identification of both known and novel isoflavone derivatives with anti-angiogenic and anti-inflammatory activity. Quantitative microflow NMR is used both to determine the structure of bioactive compounds and to quantify them for direct dose-response experiments at the microgram scale. The key advantages of this approach are (1) the microgram scale at which both biological and analytical experiments can be performed, (2) the speed and the rationality of the bioassay-guided fractionation - generic for NP extracts of diverse origin - that requires only limited sample-specific optimization and (3) the use of microflow NMR for quantification, enabling the identification and dose-response experiments with only tens of micrograms of each compound. This study demonstrates that a complete in vivo bioassay

  19. HelioScan: a software framework for controlling in vivo microscopy setups with high hardware flexibility, functional diversity and extendibility.

    Science.gov (United States)

    Langer, Dominik; van 't Hoff, Marcel; Keller, Andreas J; Nagaraja, Chetan; Pfäffli, Oliver A; Göldi, Maurice; Kasper, Hansjörg; Helmchen, Fritjof

    2013-04-30

    Intravital microscopy such as in vivo imaging of brain dynamics is often performed with custom-built microscope setups controlled by custom-written software to meet specific requirements. Continuous technological advancement in the field has created a need for new control software that is flexible enough to support the biological researcher with innovative imaging techniques and provide the developer with a solid platform for quickly and easily implementing new extensions. Here, we introduce HelioScan, a software package written in LabVIEW, as a platform serving this dual role. HelioScan is designed as a collection of components that can be flexibly assembled into microscope control software tailored to the particular hardware and functionality requirements. Moreover, HelioScan provides a software framework, within which new functionality can be implemented in a quick and structured manner. A specific HelioScan application assembles at run-time from individual software components, based on user-definable configuration files. Due to its component-based architecture, HelioScan can exploit synergies of multiple developers working in parallel on different components in a community effort. We exemplify the capabilities and versatility of HelioScan by demonstrating several in vivo brain imaging modes, including camera-based intrinsic optical signal imaging for functional mapping of cortical areas, standard two-photon laser-scanning microscopy using galvanometric mirrors, and high-speed in vivo two-photon calcium imaging using either acousto-optic deflectors or a resonant scanner. We recommend HelioScan as a convenient software framework for the in vivo imaging community.

  20. In vivo lipid saturation study of C. elegans using quantitative broadband coherent anti-Stokes Raman imaging (Conference Presentation)

    Science.gov (United States)

    Littleton, Bradley; Kavanagh, Thomas; Nie, Yu; Abbate, Vincenzo; Hylands, Peter; Sturzenbaum, Stephen; Richards, David

    2016-03-01

    In vivo lipid saturation maps of microscopic nematodes (Caenorhabditis elegans) have been produced using our novel Spectral Interferometric Polarisation Coherent anti-Stokes Raman Scattering (SIP-CARS) imaging technique. This technique employs simple passive polarisation optics and a balanced homodyne detection scheme to exploit symmetries in the CARS polarisation response resulting in the complete cancellation of the non-resonant background (NRB) and real component of the CARS signal (with no prior or post assumptions as regards to their form). The remaining imaginary component of the CARS response is linear with analyte concentration and directly relatable to the spontaneous Raman spectrum [1]. Furthermore, the resonant CARS signal is interferometrically amplified by the non-resonant response, a necessity for rapid imaging at biologically relevant powers [2]. This technique permits acquisition of a broad NRB-free spectrum, in excess of 1800cm-1, in a single exposure at each pixel. This allows simultaneous determination of lipid droplet saturation, from the fingerprint region, and lipid order, from the C-H stretch region from which maps can be readily constructed. Additionally exploiting the dispersive nature of our signal collection two-photon autofluorescence can be isolated and images subsequently produced. We have successfully applied this technique to identify differences in lipid saturation distributions in selective C. elegans mutants and demonstrated that the technique is sufficiently sensitive to detect the effects of lipid metabolism altering drugs on wild type C. elegans. [1] Littleton et al, Phys Rev Lett, 111, 103902 (2013) [2] Parekh et al, Biophys J, 99, 2695-2704 (2010)

  1. Low cost quantitative digital imaging as an alternative to qualitative in vivo bioassays for analysis of active aflatoxin B1.

    Science.gov (United States)

    Rasooly, Reuven; Do, Paula M; Hernlem, Bradley J

    2016-06-15

    Aflatoxin B1 (AFB1) producing fungi contaminate food and feed and are a major health concern. To minimize the sources and incidence of AFB1 illness there is a need to develop affordable, sensitive mobile devices for detection of active AFB1. In the present study we used a low cost fluorescence detector and describe two quantitative assays for detection of detoxified and active AFB1 demonstrating that AFB1 concentration can be measured as intensity of fluorescence. When the assay plate containing increasing concentrations of AFB1 is illuminated with a 366 nm ultraviolet lamp, AFB1 molecules absorb photons and emit blue light with peak wavelength of 432 nm. The fluorescence intensity increased in dose dependent manner. However, this method cannot distinguish between active AFB1 which poses a threat to health, and the detoxified AFB1 which exhibits no toxicity. To measure the toxin activity, we used a cell based assay that makes quantification more robust and is capable of detecting multiple samples simultaneously. It is an alternative to the qualitative duckling bioassay which is the "gold-standard" assay currently being used for quantitative analysis of active AFB1. AFB1 was incubated with transduced Vero cells expressing the green fluorescence protein (GFP) gene. After excitation with blue light at 475 nm, cells emitted green light with emission peak at 509 nm. The result shows that AFB1 inhibits protein expression in a concentration dependent manner resulting in proportionately less GFP fluorescence in cells exposed to AFB1. The result also indicates strong positive linear relationship with R(2)=0.90 between the low cost CCD camera and a fluorometer, which costs 100 times more than a CCD camera. This new analytical method for measuring active AFB1 is low in cost and combined with in vitro assay, is quantitative. It also does not require the use of animals and may be useful especially for laboratories in regions with limited resources.

  2. A quantitative in-vivo MR imaging study of brain dehydration in diabetic rats and rats treated with peptide hormones.

    Science.gov (United States)

    Haraldseth, O; Jones, R A; Skottner, A

    1997-01-01

    The main aim of the study was to evaluate the combination of quantitative diffusion, T2 and Magnetisation Transfer Imaging of brain water homeostasis using untreated diabetes as an animal model of brain dehydration. In addition, experimental groups of diabetic rats treated with insulin and insulin-like growth factor (IGF-I) and normal rats treated with IGF-I and growth hormone were studied using the same MR imaging protocol. Untreated diabetes caused weight reduction and an increase in water intake, indicating a general body dehydration linked to chronic blood hyperosmolarity. In the investigated cortical gray matter untreated diabetes caused a significant reduction in the apparent diffusion coefficient of water (ADC) and an increase in T2 relaxtivity (R2) when compared to a control group. No significant changes were observed for the calculated magnetisation transfer parameters Kfor and T1sat. Both ADC and R2 normalized after appropriate insulin treatment whereas only ADC was normalized after IGF-I treatment. IGF-I treatment of normal rats caused significantly higher rate of increase in body weight compared to normal controls. There were, however, no significant changes in ADC, R2 nor the magnetisation transfer parameters measured in the cortical gray matter of the IGF-I treated normal rats. In conclusion, we found that changes in brain water homeostasis during diabetes were detected by quantitative MR imaging, and that the dehydration induced by diabetes was normalized by insulin treatment but not by IGF-I.

  3. Quantitative Liver Function Tests: A Realizable Goal?

    Directory of Open Access Journals (Sweden)

    Denis J Morgan

    1991-01-01

    Full Text Available A variety of tests has been used to assess liver function and predict hepatic functional reserve in patients with liver disease. These tests comprise clinical assessment, simple biochemical measurements and so-called ‘quantitative’ tests of liver function, ie, elimination rate measurements of exogenous markers such as drugs and other compounds. So far no single test or group of tests has proved to be a sufficiently sensitive and accurate measure of overall hepatic function across the whole spectrum of liver disease. This may he due to diversity in the hepatic handling of these compounds and in changes in architecture, hemodynamics and cell function in liver disease. The absence of a satisfactory test emphasizes the value of clinical assessments (eg, the Child-Turcotte or Child-Pugh classifications, because of their relative simplicity.

  4. In-Vivo functional optical-resolution photoacoustic microscopy with stimulated Raman scattering fiber-laser source.

    Science.gov (United States)

    Hajireza, Parsin; Forbrich, Alexander; Zemp, Roger

    2014-02-01

    In this paper a multi-wavelength optical-resolution photoacoustic microscopy (OR-PAM) system using stimulated Raman scattering is demonstrated for both phantom and in vivo imaging. A 1-ns pulse width ytterbium-doped fiber laser is coupled into a single-mode polarization maintaining fiber. Discrete Raman-shifted wavelength peaks extending to nearly 800 nm are generated with pulse energies sufficient for OR-PAM imaging. Bandpass filters are used to select imaging wavelengths. A dual-mirror galvanometer system was used to scan the focused outputs across samples of carbon fiber networks, 200μm dye-filled tubes, and Swiss Webster mouse ears. Photoacoustic signals were collected in transmission mode and used to create maximum amplitude projection C-scan images. Double dye experiments and in vivo oxygen saturation estimation confirmed functional imaging potential.

  5. Slam haplotypes modulate the response to lipopolysaccharide in vivo through control of NKT cell number and function.

    Science.gov (United States)

    Aktan, Idil; Chant, Alan; Borg, Zachary D; Damby, David E; Leenstra, Paige C; Lilley, Graham W J; Lilley, Graham W G; Petty, Joseph; Suratt, Benjamin T; Teuscher, Cory; Wakeland, Edward K; Poynter, Matthew E; Boyson, Jonathan E

    2010-07-01

    CD1d-restricted NKT cells make up an innate-like T cell subset that plays a role in amplifying the response of innate immune leukocytes to TLR ligands. The Slam locus contains genes that have been implicated in innate and adaptive immune responses. In this study, we demonstrate that divergent Slam locus haplotypes modulate the response of macrophages to the TLR4 ligand LPS through their control of NKT cell number and function. In response to LPS challenge in vivo, macrophage TNF production in Slam haplotype-2(+) 129S1/SvImJ and 129X1/SvJ mice was significantly impaired in comparison with macrophage TNF production in Slam haplotype-1(+) C57BL/6J mice. Although no cell-intrinsic differences in macrophage responses to LPS were observed between strains, 129 mice were found to be deficient in liver NKT cell number, in NKT cell cytokine production in response to the CD1d ligand alpha-galactosylceramide, and in NKT cell IFN-gamma production after LPS challenge in vivo. Using B6.129c1 congenic mice and adoptive transfer, we found that divergent Slam haplotypes controlled the response to LPS in vivo, as well as the diminished NKT cell number and function, and that these phenotypes were associated with differential expression of signaling lymphocytic activation molecule family receptors on NKT cells. These data suggest that the polymorphisms that distinguish two Slam haplotypes significantly modulate the innate immune response in vivo through their effect on NKT cells.

  6. Dynamic contrast optical coherence tomography: quantitative measurement of microvascular transit-time distributions in vivo (Conference Presentation)

    Science.gov (United States)

    Merkle, Conrad W.; Srinivasan, Vivek J.

    2016-03-01

    Transit time is a fundamental microcirculatory parameter that is critical in determining oxygen delivery from capillaries to surrounding tissue. Recently, it was demonstrated theoretically that capillary transit-time heterogeneity potentially leads to non-uniform oxygen extraction in micro-domains. However, in spite of its importance, capillary transit-time distribution has been challenging to quantify comprehensively and efficiently at the microscopic level. Here, we introduce a method, called Dynamic Contrast Optical Coherence Tomography (DyC-OCT), based on dynamic cross-sectional OCT imaging of the kinetics of an intravascular tracer during its passage through the field-of-view. DyC-OCT is used to quantitatively measure the transit-time distribution in microvascular networks in cross-section at the single-capillary level. Transit-time metrics are derived from analysis of the temporal characteristics of the dynamic scattering signal, related to tracer concentration, using indicator-dilution theory. Since DyC-OCT does not require calibration of the optical focus, quantitative accuracy is achieved even deep in highly scattering brain tissue where the focal spot degrades. After direct validation of DyC-OCT against the dilution curves measured using a fluorescent plasma label in the surface pial vessels of a mouse brain, imaged through a thinned-skull, glass coverslip-reinforced cranial window, the laminar transit-time distribution was investigated in microvasculature across the entire depth of the mouse somatosensory cortex. Laminar trends were identified, with the earliest transit times in the middle cortical layers, and the lowest heterogeneity in cortical layer 4. The new DyC-OCT technique affords a novel perspective of microvascular networks, with the unique capability of performing simultaneous measurements of transit-time distributions across cortical laminae.

  7. Molecular advances in reporter genes: the need to witness the function of stem cells in failing heart in vivo.

    Science.gov (United States)

    Agostini, Silvia; Recchia, Fabio A; Lionetti, Vincenzo

    2012-06-01

    Stem cells possess the ability to terminally differentiate in cell phenotypes belonging to several different lineages. Over the last decade, transplant of adult stem cells into the injuried myocardium has been widely studied as a revolutionary approach to promote the non-pharmacological improvement or replacement of the lost function. In spite of the tantalizing perspectives and controversial results, several questions about the viability and biology of transplanted stem cells in the beating heart still remain unanswered, mostly because of the current technological limitations. Recent advances in bio- and nano-technology are allowing the development of molecular probes for imaging thus providing a better understanding of stem cells physiology and fate in vivo. Reporter gene based molecular imaging is a high-throughput and sensitive tool used to unscramble over time the mechanisms underlying cell-induced myocardial repair in vivo. To date, the employed reporter genes have been exogenous (proteins which are expressed after gene engineering), or endogenous (detected by tracer substrates). This review will highlight current and outstanding experimental investigations, which are developing new probes to monitor the fate of stem cells transplanted in failing myocardium in vivo.

  8. In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation.

    Science.gov (United States)

    Butko, Margaret T; Savas, Jeffrey N; Friedman, Beth; Delahunty, Claire; Ebner, Ford; Yates, John R; Tsien, Roger Y

    2013-02-19

    Postnatal bilateral whisker trimming was used as a model system to test how synaptic proteomes are altered in barrel cortex by sensory deprivation during synaptogenesis. Using quantitative mass spectrometry, we quantified more than 7,000 synaptic proteins and identified 89 significantly reduced and 161 significantly elevated proteins in sensory-deprived synapses, 22 of which were validated by immunoblotting. More than 95% of quantified proteins, including abundant synaptic proteins such as PSD-95 and gephyrin, exhibited no significant difference under high- and low-activity rearing conditions, suggesting no tissue-wide changes in excitatory or inhibitory synaptic density. In contrast, several proteins that promote mature spine morphology and synaptic strength, such as excitatory glutamate receptors and known accessory factors, were reduced significantly in deprived synapses. Immunohistochemistry revealed that the reduction in SynGAP1, a postsynaptic scaffolding protein, was restricted largely to layer I of barrel cortex in sensory-deprived rats. In addition, protein-degradation machinery such as proteasome subunits, E2 ligases, and E3 ligases, accumulated significantly in deprived synapses, suggesting targeted synaptic protein degradation under sensory deprivation. Importantly, this screen identified synaptic proteins whose levels were affected by sensory deprivation but whose synaptic roles have not yet been characterized in mammalian neurons. These data demonstrate the feasibility of defining synaptic proteomes under different sensory rearing conditions and could be applied to elucidate further molecular mechanisms of sensory development.

  9. Quantitative Evaluation of Bioorthogonal Chemistries for Surface Functionalization of Nanoparticles

    DEFF Research Database (Denmark)

    Feldborg, Lise Nørkjær; Jølck, Rasmus Irming; Andresen, Thomas Lars

    2012-01-01

    We present here a highly efficient and chemoselective liposome functionalization method based on oxime bond formation between a hydroxylamine and an aldehyde-modified lipid component. We have conducted a systematic and quantitative comparison of this new approach with other state-of-the-art...... affinity between the peptide and the liposome surface. These studies demonstrate the importance of hoosing the correct chemistry in order to obtain a quantitative surface functionalization of liposomes....

  10. In vivo biodistribution and toxicology of functionalized nano-graphene oxide in mice after oral and intraperitoneal administration.

    Science.gov (United States)

    Yang, Kai; Gong, Hua; Shi, Xiaoze; Wan, Jianmei; Zhang, Youjiu; Liu, Zhuang

    2013-04-01

    Graphene oxide (GO) and its functionalized derivatives have attracted great attention in biomedicine in recent years. A number of groups including ours have studied the in vivo behaviors of functionalized nano-graphene after intravenous injection or inhalation, and uncovered the surface coating & size dependent biodistribution and toxicology profiles for this type of nanomaterials. However, the fate of GO derivatives in animals after oral feeding and intraperitoneal (i.p.) injection, which are two other major drug administration routes, remain unclear. Therefore, in this work, we sought to systematically investigate in vivo biodistribution and potential toxicity of as-made GO and a number of polyethylene glycol (PEG) functionalized GO derivatives with different sizes and surface coatings, after oral and intraperitoneal administration at high doses. It is found that (125)I labeled PEGylated GO derivatives show no obvious tissue uptake via oral administration, indicating the rather limited intestinal adsorption of those nanomaterials. In contrast, high accumulation of PEGyalted GO derivatives, but not as-made GO, in the reticuloendothelial (RES) system including liver and spleen is observed after i.p. injection. Further investigations based on histological examination of organ slices and hematological analysis discover that although GO and PEGylated GO derivatives would retain in the mouse body over a long period of time after i.p. injection, their toxicity to the treated animals is insignificant. Our work is an important fundamental study that offers a deeper understanding of in vivo behaviors and toxicology of functionalized nano-graphene in animals, depending on their different administration routes.

  11. A semi-quantitative RT-PCR method to measure the in vivo effect of dietary conjugated linoleic acid on porcine muscle PPAR gene expression

    Directory of Open Access Journals (Sweden)

    Meadus W.J.

    2003-01-01

    Full Text Available Conjugated linoleic acid (CLA can activate (in vitro the nuclear transcription factors known as the peroxisome proliferators activated receptors (PPAR. CLA was fed at 11 g CLA/kg of feed for 45d to castrated male pigs (barrows to better understand long term effects of PPAR activation in vivo. The barrows fed CLA had lean muscle increased by 3.5% and overall fat reduced by 9.2% but intramuscular fat (IMF % was increased by 14% (P < 0.05. To measure the effect of long term feeding of CLA on porcine muscle gene expression, a semi-quantitative RT-PCR method was developed using cDNA normalized against the housekeeping genes cyclophilin and &bgr;-actin. This method does not require radioactivity or expensive PCR instruments with real-time fluorescent detection. PPAR&ggr; and the PPAR responsive gene AFABP but not PPAR&agr; were significantly increased (P < 0.05 in the CLA fed pig’s muscle. PPAR&agr; and PPAR&ggr; were also quantitatively tested for large differences in gene expression by western blot analysis but no significant difference was detected at this level. Although large differences in gene expression of the PPAR transcriptional factors could not be confirmed by western blotting techniques. The increased expression of AFABP gene, which is responsive to PPAR transcriptional factors, confirmed that dietary CLA can induce a detectable increase in basal PPAR transcriptional activity in the live animal.

  12. Optical coherence tomography based microangiography for quantitative monitoring of structural and vascular changes in a rat model of acute uveitis in vivo: a preliminary study

    Science.gov (United States)

    Choi, Woo June; Pepple, Kathryn L.; Zhi, Zhongwei; Wang, Ruikang K.

    2015-01-01

    Uveitis models in rodents are important in the investigation of pathogenesis in human uveitis and the development of appropriate therapeutic strategies for treatment. Quantitative monitoring of ocular inflammation in small animal models provides an objective metric to assess uveitis progression and/or therapeutic effects. We present a new application of optical coherence tomography (OCT) and OCT-based microangiography (OMAG) to a rat model of acute anterior uveitis induced by intravitreal injection of a killed mycobacterial extract. OCT/OMAG is used to provide noninvasive three-dimensional imaging of the anterior segment of the eyes prior to injection (baseline) and two days post-injection (peak inflammation) in rats with and without steroid treatments. OCT imaging identifies characteristic structural and vascular changes in the anterior segment of the inflamed animals when compared to baseline images. Characteristics of inflammation identified include anterior chamber cells, corneal edema, pupillary membranes, and iris vasodilation. In contrast, no significant difference from the control is observed for the steroid-treated eye. These findings are compared with the histology assessment of the same eyes. In addition, quantitative measurements of central corneal thickness and iris vessel diameter are determined. This pilot study demonstrates that OCT-based microangiography promises to be a useful tool for the assessment and management of uveitis in vivo.

  13. Quantitation of renal function using radioisotopic techniques.

    Science.gov (United States)

    O'Malley, J P; Ziessman, H A

    1993-03-01

    Radioisotopic methods are practical for clinical use because they do not require continuous intravenous infusion or urine collection. This obviously is of great advantage in infants and small children, in whom accurate urine collection is difficult, but the techniques apply to adults as well. The ability to determine individual kidney function is a major benefit. Accuracies of the radioisotopic techniques vary but generally are within clinically acceptable ranges. The need for accuracy and reproducibility can be balanced with the desire for speed and convenience when choosing among the different techniques. Methods that use plasma sampling provide greater accuracy and are recommended in cases of severe dysfunction, whereas methods such as Gates' camera method, which eliminates plasma samples, can be completed in minutes. Radioisotopic techniques are most useful in the ranges of mild to moderately decreased function, in which serum creatinine concentration is nondiagnostic, and although they are much less accurate at markedly low renal function levels, so is 24-hour creatinine clearance. In conclusion, radiopharmaceutical agents offer a wide array of possible techniques for simple, accurate, and noninvasive measurement of global as well as individual GFR and ERPF.

  14. In vivo quantitative visualization of hypochlorous acid in the liver using a novel selective two-photon fluorescent probe

    Science.gov (United States)

    Wang, Haolu; Jayachandran, Aparna; Gravot, Germain; Liang, Xiaowen; Thorling, Camilla A.; Zhang, Run; Liu, Xin; Roberts, Michael S.

    2016-11-01

    Hypochlorous acid (HOCl) plays a vital role in physiological events and diseases. During hepatic ischemia-reperfusion (I/R) injury, HOCl is generated by neutrophils and diffuses into hepatocytes, causing oxidant stress-mediated injury. Although many probes have been developed to detect HOCl, most were difficult to be distinguished from endogenous fluorophores in intravital imaging and only can be employed under one-photon microscopy. A novel iridium(III) complex-based ferrocene dual-signaling chemosensor (Ir-Fc) was designed and synthesized. Ir-Fc exhibited a strong positive fluorescent response only in the presence of HOCl, whereas negligible fluorescent signals were observed upon the additions of other reactive oxygen/nitrogen species and metal ions. There was a good linear relationship between probe responsive fluorescent intensity and HOCl concentration. Ir-Fc was then intravenously injected into BALB/c mice at the final concentration of 50 μM and the mouse livers were imaged using multiphoton microscopy (MPM). In the I/R liver, reduced autofluorescence was detected by MPM, indicating the hepatocyte necrosis. Remarkable enhancement of red fluorescence was observed in hepatocytes with decreased autofluorescence, indicating the reaction of Ir-Fc with endogenous HOCl molecules. The cellular concentration of HOCl was first calculated based on the intensity of MPM images. No obvious toxic effects were observed in histological examination of major organs after Ir-Fc injection. In summary, Ir-Fc has low cytotoxicity, high specificity to HOCl, and rapid "off-on" fluorescence. It is suitable for dynamic quantitatively monitoring HOCl generation using MPM at the cellular level. This technique can be readily extended to examination of liver diseases and injury.

  15. Computer aided prognosis for cell death categorization and prediction in vivo using quantitative ultrasound and machine learning techniques.

    Science.gov (United States)

    Gangeh, M J; Hashim, A; Giles, A; Sannachi, L; Czarnota, G J

    2016-12-01

    At present, a one-size-fits-all approach is typically used for cancer therapy in patients. This is mainly because there is no current imaging-based clinical standard for the early assessment and monitoring of cancer treatment response. Here, the authors have developed, for the first time, a complete computer-aided-prognosis (CAP) system based on multiparametric quantitative ultrasound (QUS) spectroscopy methods in association with texture descriptors and advanced machine learning techniques. This system was used to noninvasively categorize and predict cell death levels in fibrosarcoma mouse tumors treated using ultrasound-stimulated microbubbles as novel endothelial-cell radiosensitizers. Sarcoma xenograft tumor-bearing mice were treated using ultrasound-stimulated microbubbles, alone or in combination with x-ray radiation therapy, as a new antivascular treatment. Therapy effects were assessed at 2-3, 24, and 72 h after treatment using a high-frequency ultrasound. Two-dimensional spectral parametric maps were generated using the power spectra of the raw radiofrequency echo signal. Subsequently, the distances between "pretreatment" and "post-treatment" scans were computed as an indication of treatment efficacy, using a kernel-based metric on textural features extracted from 2D parametric maps. A supervised learning paradigm was used to either categorize cell death levels as low, medium, or high using a classifier, or to "continuously" predict the levels of cell death using a regressor. The developed CAP system performed at a high level for the classification of cell death levels. The area under curve of the receiver operating characteristic was 0.87 for the classification of cell death levels to both low/medium and medium/high levels. Moreover, the prediction of cell death levels using the proposed CAP system achieved a good correlation (r = 0.68,  p course of therapy to enable switching to more efficacious treatments.

  16. Development of a Novel, Highly Quantitative In Vivo Model for the Study of Biofilm-Impaired Cutaneous Wound Healing

    Science.gov (United States)

    2011-01-01

    established on microbiologically naıve tissues (such as in endocarditis or cystic fibrosis ). Whether on damaged heart valves or poorly functioning respiratory...addition to the classic tenets of hypoxia, ischemia-reperfusion, and intrinsic host disease , then, bac- terial biofilms hold considerable promise as a fourth...Stoodley L, Costerton JW, Stoodley P. Bacterial bio films: from the natural environment to infectious diseases . Nat Rev Microbiol 2004; 2: 95 108. 2. Parsek

  17. Utilization of quantitative in vivo pharmacology approaches to assess combination effects of everolimus and irinotecan in mouse xenograft models of colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Erica L Bradshaw-Pierce

    Full Text Available PURPOSE: The PI3K/AKT/mTOR pathway is frequently dysregulated in cancers and inhibition of mTOR has demonstrated the ability to modulate pro-survival pathways. As such, we sought to determine the ability of the mTOR inhibitor everolimus to potentiate the antitumor effects of irinotecan in colorectal cancer (CRC. EXPERIMENTAL DESIGN: The combinatorial effects of everolimus and irinotecan were evaluated in vitro and in vivo in CRC cell lines harboring commonly found mutations in PIK3CA, KRAS and/or BRAF. Pharmacokinetically-directed dosing protocols of everolimus and irinotecan were established and used to assess the in vivo antitumor effects of the agents. At the end of treatment, 3-6 tumors per treatment arm were harvested for biomarker analysis by NMR metabolomics. RESULTS: Everolimus and irinotecan/SN38 demonstrated synergistic anti-proliferative effects in multiple CRC cell lines in vitro. Combination effects of everolimus and irinotecan were determined in CRC xenograft models using clinically-relevant dosing protocols. Everolimus demonstrated significant tumor growth inhibition alone and when combined with irinotecan in HT29 and HCT116 tumor xenografts. Metabolomic analysis showed that HT29 tumors were more metabolically responsive than HCT116 tumors. Everolimus caused a decrease in glycolysis in both tumor types whilst irinotecan treatment resulted in a profound accumulation of lipids in HT29 tumors indicating a cytotoxic effect. CONCLUSIONS: Quantitative analysis of tumor growth and metabolomic data showed that the combination of everolimus and irinotecan was more beneficial in the BRAF/PIK3CA mutant HT29 tumor xenografts, which had an additive effect, than the KRAS/PIK3CA mutant HCT116 tumor xenografts, which had a less than additive effect.

  18. Fucoidan can function as an adjuvant in vivo to enhance dendritic cell maturation and function and promote antigen-specific T cell immune responses.

    Directory of Open Access Journals (Sweden)

    Jun-O Jin

    Full Text Available Fucoidan, a sulfated polysaccharide purified from brown algae, has a variety of immune-modulation effects, including promoting antigen uptake and enhancing anti-viral and anti-tumor effects. However, the effect of fucoidan in vivo, especially its adjuvant effect on in vivo anti-tumor immune responses, was not fully investigated. In this study, we investigated the effect of fucoidan on the function of spleen dendritic cells (DCs and its adjuvant effect in vivo. Systemic administration of fucoidan induced up-regulation of CD40, CD80 and CD86 expression and production of IL-6, IL-12 and TNF-α in spleen cDCs. Fucoidan also promoted the generation of IFN-γ-producing Th1 and Tc1 cells in an IL-12-dependent manner. When used as an adjuvant in vivo with ovalbumin (OVA antigen, fucoidan promoted OVA-specific antibody production and primed IFN-γ production in OVA-specific T cells. Moreover, fucoidan enhanced OVA-induced up-regulation of MHC class I and II on spleen cDCs and strongly prompted the proliferation of OVA-specific CD4 and CD8 T cells. Finally, OVA immunization with fucoidan as adjuvant protected mice from the challenge with B16-OVA tumor cells. Taken together, these results suggest that fucoidan can function as an adjuvant to induce Th1 immune response and CTL activation, which may be useful in tumor vaccine development.

  19. Quantitation of dopamine transporter blockade by methylphenidate: first in vivo investigation using [{sup 123}I]FP-CIT and a dedicated small animal SPECT

    Energy Technology Data Exchange (ETDEWEB)

    Nikolaus, Susanne; Wirrwar, Andreas; Antke, Christina; Arkian, Shahram; Mueller, Hans-Wilhelm; Larisch, Rolf [Heinrich-Heine University, Clinic of Nuclear Medicine, Duesseldorf (Germany); Schramm, Nils [Research Center Juelich, Central Laboratory for Electronics, Juelich (Germany)

    2005-03-01

    The aim of this study was to investigate the feasibility of assessing dopamine transporter binding after treatment with methylphenidate in the rat using a recently developed high-resolution small animal single-photon emission computed tomograph (TierSPECT) and [{sup 123}I]FP-CIT. [{sup 123}I]FP-CIT was administered intravenously 1 h after intraperitoneal injection of methylphenidate (10 mg/kg) or vehicle. Animals underwent scanning 2 h after radioligand administration. The striatum was identified by superimposition of [{sup 123}I]FP-CIT scans with bone metabolism and perfusion scans obtained with {sup 99m}Tc-DPD and {sup 99m}Tc-tetrofosmin, respectively. As these tracers do not pass the blood-brain barrier, their distribution permits the identification of extracerebral anatomical landmarks such as the orbitae and the harderian glands. The cerebellum was identified by superimposing [{sup 123}I]FP-CIT scans with images of brain perfusion obtained with {sup 99m}Tc-HMPAO. Methylphenidate-treated animals and vehicle-treated animals yielded striatal equilibrium ratios (V''{sub 3}) of 0.24{+-}0.26 (mean {+-} SD) and 1.09{+-}0.42, respectively (ttest, two-tailed, p<0.0001). Cortical V''{sub 3} values amounted to 0.05{+-}0.28 (methylphenidate) and 0.3{+-}0.39 (saline, p=0.176). This first in vivo study of rat dopamine transporter binding after pre-treatment with methylphenidate showed a mean reduction of 78% in striatal [{sup 123}I]FP-CIT accumulation. The results can be interpreted in terms of a pharmacological blockade in the rat striatum and show that in vivo quantitation of dopamine transporter binding is feasible with [{sup 123}I]FP-CIT and the TierSPECT. This may be of future relevance for in vivo investigations on rat models of attention deficit/hyperactivity disorder. Furthermore, our findings suggest that investigations in other animal models, e.g. of Parkinson's and Huntington's disease, may be feasible using SPECT radioligands and

  20. Surface-Functionalized Nanoparticles by Olefin Metathesis: A Chemoselective Approach for In Vivo Characterization of Atherosclerosis Plaque.

    Science.gov (United States)

    Salinas, Beatriz; Ruiz-Cabello, Jesús; Lechuga-Vieco, Ana V; Benito, Marina; Herranz, Fernando

    2015-07-13

    The use of click chemistry reactions for the functionalization of nanoparticles is particularly useful to modify the surface in a well-defined manner and to enhance the targeting properties, thus facilitating clinical translation. Here it is demonstrated that olefin metathesis can be used for the chemoselective functionalization of iron oxide nanoparticles with three different examples. This approach enables, in one step, the synthesis and functionalization of different water-stable magnetite-based particles from oleic acid-coated counterparts. The surface of the nanoparticles was completely characterized showing how the metathesis approach introduces a large number of hydrophilic molecules on their coating layer. As an example of the possible applications of these new nanocomposites, a focus was taken on atherosclerosis plaques. It is also demonstrated how the in vitro properties of one of the probes, particularly its Ca(2+) -binding properties, mediate their final in vivo use; that is, the selective accumulation in atherosclerotic plaques. This opens promising new applications to detect possible microcalcifications associated with plaque vulnerability. The accumulation of the new imaging tracers is demonstrated by in vivo magnetic resonance imaging of carotids and aorta in the ApoE(-/-) mouse model and the results were confirmed by histology.

  1. A functional genomics approach using radiation-induced changes in gene expression to study low dose radiation effects in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Fornace, Jr, A J

    2007-03-03

    Abstract for final report for project entitled A functional genomics approach using radiation-induced changes in gene expression to study low dose radiation effects in vitro and in vivo which has been supported by the DOE Low Dose Radiation Research Program for approximately 7 years. This project has encompassed two sequential awards, ER62683 and then ER63308, in the Gene Response Section in the Center for Cancer Research at the National Cancer Institute. The project was temporarily suspended during the relocation of the Principal Investigators laboratory to the Dept. of Genetics and Complex Diseases at Harvard School of Public Health at the end of 2004. Remaining support for the final year was transferred to this new site later in 2005 and was assigned the DOE Award Number ER64065. The major aims of this project have been 1) to characterize changes in gene expression in response to low-dose radiation responses; this includes responses in human cells lines, peripheral blood lymphocytes (PBL), and in vivo after human or murine exposures, as well as the effect of dose-rate on gene responses; 2) to characterize changes in gene expression that may be involved in bystander effects, such as may be mediated by cytokines and other intercellular signaling proteins; and 3) to characterize responses in transgenic mouse models with relevance to genomic stability. A variety of approaches have been used to study transcriptional events including microarray hybridization, quantitative single-probe hybridization which was developed in this laboratory, quantitative RT-PCR, and promoter microarray analysis using genomic regulatory motifs. Considering the frequent responsiveness of genes encoding cytokines and related signaling proteins that can affect cellular metabolism, initial efforts were initiated to study radiation responses at the metabolomic level and to correlate with radiation-responsive gene expression. Productivity includes twenty-four published and in press manuscripts

  2. In vivo imaging of dopamine transporter function in rat striatum using pinhole SPECT and 123I-beta-CIT coregistered with small animal MRI

    CERN Document Server

    Dierkes, K

    2001-01-01

    The aim of this study was to establish in vivo imaging of dopamine transporter function in a small animal model of Parkinson's disease using pinhole SPECT and 123I labeled beta-CIT. Since functional imaging of small animals can hardly be interpreted without localization to related anatomical structures, MRI-SPECT coregistration secondly was established as an inexpensive tool for in vivo monitoring of physiological and pathological alterations in striatal dopamine transporters using beta-CIT as an specific radionuclear ligand.

  3. In vivo optical imaging of human retinal capillary networks using speckle variance optical coherence tomography with quantitative clinico-histological correlation.

    Science.gov (United States)

    Chan, Geoffrey; Balaratnasingam, Chandrakumar; Xu, Jing; Mammo, Zaid; Han, Sherry; Mackenzie, Paul; Merkur, Andrew; Kirker, Andrew; Albiani, David; Sarunic, Marinko V; Yu, Dao-Yi

    2015-07-01

    Retinal capillary networks are critically linked to neuronal health and disease. The ability to perform accurate in vivo examination of human retinal capillary networks is therefore valuable for studying mechanisms that govern retinal homeostasis and retinal vascular diseases. Speckle variance optical coherence tomography (svOCT) is a non-invasive imaging technique that has the capacity to provide angiographic information about the retinal circulation. The application of this technology for studying human retinal capillary networks however has not been validated in a quantifiable manner. We use a custom-built svOCT device to qualitatively and quantitatively study the various capillary networks in the human perifovea. Capillary networks corresponding to the nerve fibre layer (NFL), the retinal ganglion cell/superficial inner plexiform layer (RGC/sIPL), the deep inner plexiform layer/superficial inner nuclear layer (dIPL/sINL) and the deep inner nuclear layer (dINL) are imaged in 9 normal human subjects. Measurements of capillary diameter and capillary density are made from each of these networks and results are compared to post-mortem histological data acquired with confocal scanning laser microscopy. Additionally, retinal capillary measurements from high-resolution fundus fluorescein angiogram (FA) are directly compared with svOCT images from 6 eyes. We demonstrate that svOCT images of capillary networks are morphologically comparable to microscopic images of histological specimens. Similar to histological images in svOCT images, the capillaries in the NFL network run parallel to the direction of RGC axons while capillaries in the dINL network comprise a planar configuration with multiple closed loops. Capillaries in remaining networks are convoluted with a complex three-dimensional architecture. We demonstrate that there is no significant difference in capillary density measurements between svOCT and histology images for all networks. Capillary diameter was

  4. Selective Delivery of an Anticancer Drug with Aptamer-Functionalized Liposomes to Breast Cancer Cells in Vitro and in Vivo.

    Science.gov (United States)

    Xing, Hang; Tang, Li; Yang, Xujuan; Hwang, Kevin; Wang, Wendan; Yin, Qian; Wong, Ngo Yin; Dobrucki, Lawrence W; Yasui, Norio; Katzenellenbogen, John A; Helferich, William G; Cheng, Jianjun; Lu, Yi

    2013-10-21

    Selective targeting of cancer cells is a critical step in cancer diagnosis and therapy. To address this need, DNA aptamers have attracted significant attention as possible targeting ligands. However, while their use in targeting cancer cells in vitro has been reported, their effectiveness has rarely been established in vivo. Here we report the development of a liposomal drug delivery system for targeted anticancer chemotherapy. Liposomes were prepared containing doxorubicin as a payload, and functionalized with AS1411, a DNA aptamer with strong binding affinity for nucleolin. AS1411 aptamer-functionalized liposomes increased cellular internalization and cytotoxicity to MCF-7 breast cancer cells as compared to non-targeting liposomes. Furthermore, targeted liposomal doxorubicin improved antitumor efficacy against xenograft MCF-7 breast tumors in athymic nude mice, attributable to their enhanced tumor tissue penetration. This study suggests that AS1411 aptamer-functionalized liposomes can recognize nucleolin overexpressed on MCF-7 cell surface, and therefore enable drug delivery with high specificity and selectivity.

  5. Dendritic cell function in vivo during the steady state: a role in peripheral tolerance.

    Science.gov (United States)

    Steinman, Ralph M; Hawiger, Daniel; Liu, Kang; Bonifaz, Laura; Bonnyay, David; Mahnke, Karsten; Iyoda, Tomonori; Ravetch, Jeffrey; Dhodapkar, Madhav; Inaba, Kayo; Nussenzweig, Michel

    2003-04-01

    The avoidance of autoimmunity requires mechanisms to actively silence or tolerize self reactive T cells in the periphery. During infection, dendritic cells are not only capturing microbial antigens, but also are processing self antigens from dying cells as well as innocuous environmental proteins. Since the dendritic cells are maturing in response to microbial and other stimuli, peptides will be presented from both noxious and innocuous antigens. Therefore it would be valuable to have mechanisms whereby dendritic cells, prior to infection, establish tolerance to those self and environmental antigens that can be processed upon pathogen encounter. In the steady state, prior to acute infection and inflammation, dendritic cells are in an immature state and not fully differentiated to carry out their known roles as inducers of immunity. These immature cells are not inactive, however. They continuously circulate through tissues and into lymphoid organs, capturing self antigens as well as innocuous environmental proteins. Recent experiments have provided direct evidence that antigen-loaded immature dendritic in vivo silence T cells either by deleting them or by expanding regulatory T cells. In this way, it is proposed that the immune system overcomes at least some of the risk of developing autoimmunity and chronic inflammation. It is proposed that dendritic cells play a major role in defining immunologic self, not only centrally in the thymus but also in the periphery.

  6. A functional Tyr1306Cys variant in LARG is associated with increased insulin action in vivo.

    Science.gov (United States)

    Kovacs, Peter; Stumvoll, Michael; Bogardus, Clifton; Hanson, Robert L; Baier, Leslie J

    2006-05-01

    Diminished insulin sensitivity is a characteristic feature of type 2 diabetes. Inhibition of insulin action, resulting in reduced skeletal muscle glucose uptake, is mediated in part through stimulation of RhoA activity. One regulator of RhoA activity is leukemia-associated Rho guanine nucleotide exchange factor (LARG). The LARG gene maps to a region on chromosome 11q23-24 that shows genetic linkage to BMI and type 2 diabetes in Pima Indians. Because of its role in RhoA activation, the LARG gene was analyzed as a positional candidate gene for this linkage. Sequencing of the LARG gene and genotyping of variants identified several polymorphisms that were associated with in vivo rates of insulin-mediated glucose uptake, at both physiological and maximally stimulating insulin concentrations, among 322 nondiabetic Pima Indians who had undergone a hyperinsulinemic-euglycemic clamp. The strongest association with rate of glucose uptake was found with a Tyr1306Cys polymorphism (P LARG(Cys1306) protein had reduced activity compared with LARG(Tyr1306) protein (P LARG, through its differential activation of RhoA, increases insulin sensitivity in nondiabetic Pima Indians.

  7. Genome-scale functional characterization of Drosophila developmental enhancers in vivo.

    Science.gov (United States)

    Kvon, Evgeny Z; Kazmar, Tomas; Stampfel, Gerald; Yáñez-Cuna, J Omar; Pagani, Michaela; Schernhuber, Katharina; Dickson, Barry J; Stark, Alexander

    2014-08-01

    Transcriptional enhancers are crucial regulators of gene expression and animal development and the characterization of their genomic organization, spatiotemporal activities and sequence properties is a key goal in modern biology. Here we characterize the in vivo activity of 7,705 Drosophila melanogaster enhancer candidates covering 13.5% of the non-coding non-repetitive genome throughout embryogenesis. 3,557 (46%) candidates are active, suggesting a high density with 50,000 to 100,000 developmental enhancers genome-wide. The vast majority of enhancers display specific spatial patterns that are highly dynamic during development. Most appear to regulate their neighbouring genes, suggesting that the cis-regulatory genome is organized locally into domains, which are supported by chromosomal domains, insulator binding and genome evolution. However, 12 to 21 per cent of enhancers appear to skip non-expressed neighbours and regulate a more distal gene. Finally, we computationally identify cis-regulatory motifs that are predictive and required for enhancer activity, as we validate experimentally. This work provides global insights into the organization of an animal regulatory genome and the make-up of enhancer sequences and confirms and generalizes principles from previous studies. All enhancer patterns are annotated manually with a controlled vocabulary and all results are available through a web interface (http://enhancers.starklab.org), including the raw images of all microscopy slides for manual inspection at arbitrary zoom levels.

  8. Single cell electroporation for longitudinal imaging of synaptic structure and function in the adult mouse neocortex in vivo

    Directory of Open Access Journals (Sweden)

    Stephane ePages

    2015-04-01

    Full Text Available Longitudinal imaging studies of neuronal structures in vivo have revealed rich dynamics in dendritic spines and axonal boutons. Spines and boutons are considered to be proxies for synapses. This implies that synapses display similar dynamics. However, spines and boutons do not always bear synapses, some may contain more than one, and dendritic shaft synapses have no clear structural proxies. In addition, synaptic strength is not always accurately revealed by just the size of these structures. Structural and functional dynamics of synapses could be studied more reliably using fluorescent synaptic proteins as markers for size and function. These proteins are often large and possibly interfere with circuit development, which renders them less suitable for conventional transfection or transgenesis methods such as viral vectors, in utero electroporation and germline transgenesis. Single cell electroporation has been shown to be a potential alternative for transfection of recombinant fluorescent proteins in adult cortical neurons. Here we provide proof of principle for the use of single cell electroporation to express and subsequently image fluorescently tagged synaptic proteins over days to weeks in vivo.

  9. Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidende in rodent bioassays

    NARCIS (Netherlands)

    Paini, A.; Scholz, G.; Marin-Kuan, M.; Schilter, B.; O'Brien, J.; Bladeren, van P.J.; Rietjens, I.

    2011-01-01

    This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate w

  10. Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidende in rodent bioassays

    NARCIS (Netherlands)

    Paini, A.; Scholz, G.; Marin-Kuan, M.; Schilter, B.; O'Brien, J.; Bladeren, van P.J.; Rietjens, I.

    2011-01-01

    This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate

  11. Prostate stem cell antigen-targeted nanoparticles with dual functional properties: in vivo imaging and cancer chemotherapy

    Directory of Open Access Journals (Sweden)

    Gao X

    2012-07-01

    Full Text Available Xin Gao,1,* Yun Luo,1,* Yuanyuan Wang,1,* Jun Pang,1 Chengde Liao,2 Hanlun Lu,3 Youqiang Fang11Department of Urology, The Third Affiliated Hospital, 2Department of Radiology, The Second Affiliated Hospital, Sun Yat-Sen University, 3Materials Science Institute of Zhongshan University, Guangzhou, China*These authors contributed equally to this workBackground: We designed dual-functional nanoparticles for in vivo application using a modified electrostatic and covalent layer-by-layer assembly strategy to address the challenge of assessment and treatment of hormone-refractory prostate cancer.Methods: Core-shell nanoparticles were formulated by integrating three distinct functional components, ie, a core constituted by poly(D,L-lactic-co-glycolic acid, docetaxel, and hydrophobic superparamagnetic iron oxide nanocrystals (SPIONs, a multilayer shell formed by poly(allylamine hydrochloride and two different sized poly(ethylene glycol molecules, and a single-chain prostate stem cell antigen antibody conjugated to the nanoparticle surface for targeted delivery.Results: Drug release profiles indicated that the dual-function nanoparticles had a sustained release pattern over 764 hours, and SPIONs could facilitate the controlled release of the drug in vitro. The nanoparticles showed increased antitumor efficiency and enhanced magnetic resonance imaging in vitro through targeted delivery of docetaxel and SPIONs to PC3M cells. Moreover, in nude mice bearing PC3M xenografts, the nanoparticles provided MRI negative contrast enhancement, as well as halting and even reversing tumor growth during the 76-day study duration, and without significant systemic toxicity. The lifespan of the mice treated with these targeted dual-function nanoparticles was significantly increased (Chi-square = 22.514, P < 0.0001.Conclusion: This dual-function nanomedical platform may be a promising candidate for tumor imaging and targeted delivery of chemotherapeutic agents in vivo

  12. Development of an in vivo RNAi protocol to investigate gene function in the filarial nematode, Brugia malayi.

    Directory of Open Access Journals (Sweden)

    Chuanzhe Song

    Full Text Available Our ability to control diseases caused by parasitic nematodes is constrained by a limited portfolio of effective drugs and a paucity of robust tools to investigate parasitic nematode biology. RNA interference (RNAi is a reverse-genetics tool with great potential to identify novel drug targets and interrogate parasite gene function, but present RNAi protocols for parasitic nematodes, which remove the parasite from the host and execute RNAi in vitro, are unreliable and inconsistent. We have established an alternative in vivo RNAi protocol targeting the filarial nematode Brugia malayi as it develops in an intermediate host, the mosquito Aedes aegypti. Injection of worm-derived short interfering RNA (siRNA and double stranded RNA (dsRNA into parasitized mosquitoes elicits suppression of B. malayi target gene transcript abundance in a concentration-dependent fashion. The suppression of this gene, a cathepsin L-like cysteine protease (Bm-cpl-1 is specific and profound, both injection of siRNA and dsRNA reduce transcript abundance by 83%. In vivo Bm-cpl-1 suppression results in multiple aberrant phenotypes; worm motility is inhibited by up to 69% and parasites exhibit slow-moving, kinked and partial-paralysis postures. Bm-cpl-1 suppression also retards worm growth by 48%. Bm-cpl-1 suppression ultimately prevents parasite development within the mosquito and effectively abolishes transmission potential because parasites do not migrate to the head and proboscis. Finally, Bm-cpl-1 suppression decreases parasite burden and increases mosquito survival. This is the first demonstration of in vivo RNAi in animal parasitic nematodes and results indicate this protocol is more effective than existing in vitro RNAi methods. The potential of this new protocol to investigate parasitic nematode biology and to identify and validate novel anthelmintic drug targets is discussed.

  13. Hemo oxygenase-1 induction in vitro and in vivo can yield pancreas islet xenograft survival and improve islet function

    Institute of Scientific and Technical Information of China (English)

    CHEN Xi; SU Chang; ZHANG Zheng-yun; ZHANG Ming-jun; GU Wei-qiong; LI Xiao-ying; LI Hong-wei; ZHOU Guang-wen

    2011-01-01

    Background The induced expression of heme oxygenase-1 (HO-1) in donor islets improves allograft survival.Cobalt protoporphyrin (CoPP) could significantly enhance the expression of HO-1 mRNA and protein in rat islet safely.Our work was to study how to protect pancreatic islet xenograft by CoPP-induction.Methods Islet xenografts treated with CoPP-induction and CoPP+ Zinc protoporphyrin (ZnPP) in vitro and in vivo were randomly transplanted into murine subrenal capsule; then the graft survival time was compared by blood glucose level and pathological examination and meanwhile the interferon γ (IFN-γ),tumor necrosis factor a (TNF-α),interleukin 10 (IL-10) and IL-1β level in serum and their mRNA and HO-1 mRNA and protein expression were examined.Results Islets with CoPP-induction under low- and high-glucose stimulation exhibited much higher insulin secretion compared with other three groups.CoPP-induction could increase higher expression of HO-1 (mRNA:3.33- and 76.09-fold in vitro and in vivo; protein:2.85- and 58.72-fold).The normoglycemia time in induction groups ((14.63±1.19) and (16.88+1.64) days) was significantly longer.The pathological examination showed less lymphocyte infiltration in induction groups.The IL-10 level and its mRNA in induction groups were significantly higher.Conclusions The HO-1 induced by CoPP would significantly improve function,prolong normoglycemia time and reduce lymphocyte infiltration.Meanwhile CoPP-induction in vivo had more beneficial effects than in vitro.Its mechanism could be related to immune-modulation of IL-10.

  14. Diffusion spectrum imaging shows the structural basis of functional cerebellar circuits in the human cerebellum in vivo.

    Directory of Open Access Journals (Sweden)

    Cristina Granziera

    Full Text Available BACKGROUND: The cerebellum is a complex structure that can be affected by several congenital and acquired diseases leading to alteration of its function and neuronal circuits. Identifying the structural bases of cerebellar neuronal networks in humans in vivo may provide biomarkers for diagnosis and management of cerebellar diseases. OBJECTIVES: To define the anatomy of intrinsic and extrinsic cerebellar circuits using high-angular resolution diffusion spectrum imaging (DSI. METHODS: We acquired high-resolution structural MRI and DSI of the cerebellum in four healthy female subjects at 3T. DSI tractography based on a streamline algorithm was performed to identify the circuits connecting the cerebellar cortex with the deep cerebellar nuclei, selected brainstem nuclei, and the thalamus. RESULTS: Using in-vivo DSI in humans we were able to demonstrate the structure of the following cerebellar neuronal circuits: (1 connections of the inferior olivary nucleus with the cerebellar cortex, and with the deep cerebellar nuclei (2 connections between the cerebellar cortex and the deep cerebellar nuclei, (3 connections of the deep cerebellar nuclei conveyed in the superior (SCP, middle (MCP and inferior (ICP cerebellar peduncles, (4 complex intersections of fibers in the SCP, MCP and ICP, and (5 connections between the deep cerebellar nuclei and the red nucleus and the thalamus. CONCLUSION: For the first time, we show that DSI tractography in humans in vivo is capable of revealing the structural bases of complex cerebellar networks. DSI thus appears to be a promising imaging method for characterizing anatomical disruptions that occur in cerebellar diseases, and for monitoring response to therapeutic interventions.

  15. In vivo measurements of the triceps surae complex architecture in man: implications for muscle function.

    Science.gov (United States)

    Maganaris, C N; Baltzopoulos, V; Sargeant, A J

    1998-10-15

    1. The objectives of this study were to (1) quantify experimentally in vivo changes in pennation angle, fibre length and muscle thickness in the triceps surae complex in man in response to changes in ankle position and isometric plantarflexion moment and (2) compare changes in the above muscle architectural characteristics occurring in the transition from rest to a given isometric plantarflexion intensity with the estimations of a planimetric muscle model assuming constant thickness and straight muscle fibres. 2. The gastrocnemius medialis (GM), gastrocnemius lateralis (GL) and soleus (SOL) muscles of six males were scanned with ultrasonography at different sites along and across the muscle belly at rest and during maximum voluntary contraction (MVC) trials at ankle angles of -15 deg (dorsiflexed direction), 0 deg (neutral position), +15 deg (plantarflexed direction) and +30 deg. Additional images were taken at 80, 60, 40 and 20% of MVC at an ankle angle of 0 deg. 3. In all three muscles and all scanned sites, as ankle angle increased from -15 to +30 deg, pennation increased (by 6-12 deg, 39-67%, P MVC) and fibre length decreased (by 15-28 mm, 32-34%, P MVC). Thickness in GL and SOL increased during MVC compared with rest (by 5-7 mm, 36-47%, P 0.05) between rest and MVC. 4. At any given ankle angle the model underestimated changes in GL and SOL occurring in the transition from rest to MVC in pennation angle (by 9-12 deg, 24-38%, P architecture during contraction compared with rest.

  16. Functional role of gap junctions in cytokine-induced leukocyte adhesion to endothelium in vivo

    Science.gov (United States)

    Véliz, Loreto P.; González, Francisco G.; Duling, Brian R.; Sáez, Juan C.; Boric, Mauricio P.

    2008-01-01

    To assess the hypothesis that gap junctions (GJs) participate on leukocyte-endothelium interactions in the inflammatory response, we compared leukocyte adhesion and transmigration elicited by cytokine stimulation in the presence or absence of GJ blockers in the hamster cheek pouch and also in the cremaster muscle of wild-type (WT) and endothelium-specific connexin 43 (Cx43) null mice (Cx43e−/−). In the cheek pouch, topical tumor necrosis factor-α (TNF-α; 150 ng/ml, 15 min) caused a sustained increment in the number of leukocytes adhered to venular endothelium (LAV) and located at perivenular regions (LPV). Superfusion with the GJ blockers 18-α-glycyrrhetinic acid (AGA; 75 μM) or 18-β-glycyrrhetinic acid (50 μM) abolished the TNF-α-induced increase in LAV and LPV; carbenoxolone (75 μM) or oleamide (100 μM) reduced LAV by 50 and 75%, respectively, and LPV to a lesser extent. None of these GJ blockers modified venular diameter, blood flow, or leukocyte rolling. In contrast, glycyrrhizin (75 μM), a non-GJ blocker analog of AGA, was devoid of effect. Interestingly, when AGA was removed 90 min after TNF-α stimulation, LAV started to rise at a similar rate as in control. Conversely, application of AGA 90 min after TNF-α reduced the number of previously adhered cells. In WT mice, intrascrotal injection of TNF-α (0.5 μg/0.3 ml) increased LAV (fourfold) and LPV (threefold) compared with saline-injected controls. In contrast to the observations in WT animals, TNF-α stimulation did not increase LAV or LPV in Cx43e−/− mice. These results demonstrate an important role for GJ communication in leukocyte adhesion and transmigration during acute inflammation in vivo and further suggest that endothelial Cx43 is key in these processes. PMID:18599597

  17. Lack of CAR impacts neuronal function and cerebrovascular integrity in vivo.

    Science.gov (United States)

    Boussadia, Baddreddine; Gangarossa, Giuseppe; Mselli-Lakhal, Laila; Rousset, Marie-Claude; de Bock, Frederic; Lassere, Frederic; Ghosh, Chaitali; Pascussi, Jean-Marc; Janigro, Damir; Marchi, Nicola

    2016-09-01

    Nuclear receptors (NRs) are a group of transcription factors emerging as players in normal and pathological CNS development. Clinically, an association between the constitutive androstane NR (CAR) and cognitive impairment was proposed, however never experimentally investigated. We wished to test the hypothesis that the impact of CAR on neurophysiology and behavior is underlined by cerebrovascular-neuronal modifications. We have used CAR(-/-) C57BL/6 and wild type mice and performed a battery of behavioral tests (recognition, memory, motor coordination, learning and anxiety) as well as longitudinal video-electroencephalographic recordings (EEG). Brain cell morphology was assessed using 2-photon or electron microscopy and fluorescent immunohistochemistry. We observed recognition memory impairment and increased anxiety-like behavior in CAR(-/-) mice, while locomotor activity was not affected. Concomitantly to memory deficits, EEG monitoring revealed a decrease in 3.5-7Hz waves during the awake/exploration and sleep periods. Behavioral and EEG abnormalities in CAR(-/-) mice mirrored structural changes, including tortuous fronto-parietal penetrating vessels. At the cellular level we found reduced ZO-1, but not CLDN5, tight junction protein expression in cortical and hippocampal isolated microvessel preparations. Interestingly, the neurotoxin kainic acid, when injected peripherally, provoked a rapid onset of generalized convulsions in CAR(-/-) as compared to WT mice, supporting the hypothesis of vascular permeability. The morphological phenotype of CAR(-/-) mice also included some modifications of GFAP/IBA1 glial cells in the parenchymal or adjacent to collagen-IV(+) or FITC(+) microvessels. Neuronal defects were also observed including increased cortical NEUN(+) cell density, hippocampal granule cell dispersion and increased NPY immunoreactivity in the CA1 region in CAR(-/-) mice. The latter may contribute to the in vivo phenotype. Our results indicate that behavioral

  18. DNA-damaging activity in vivo and bacterial mutagenicity of sixteen aromatic amines and azo-derivatives, as related quantitatively to their carcinogenicity.

    Science.gov (United States)

    Parodi, S; Taningher, M; Russo, P; Pala, M; Tamaro, M; Monti-Bragadin, C

    1981-01-01

    Sixteen aromatic amines and azo-derivatives were studied. They were: benzidine; 2-acetylaminofluorene; 3'-methyl-p-dimethylaminobenzene; o-aminoazo-toluene; p-dimethylaminoazobenzene; 2,4-diamino-toluene; 4,4'-oxydianiline; 2,4-diaminoanisole; 4,4'-methylenedianiline; 2-naphthylamine; Auramine O; Rhodamine B; Ponceau MX; 1-naphthylamine; p-aminoazobenzene and aniline. The compounds were examined for their capability to induce alkaline DNA fragmentation in rat liver after treatment in vivo, for their mutagenicity in the Salmonella strains TA 98 and TA 100, for their acute toxicity and for their carcinogenicity in mice and rats. For each parameter a quantitative potency index was established, and the correlation existing amongst the different parameters investigated. Only mutagenicity in the strain TA 98 was slightly correlated with carcinogenic potency (r = 0.408). DNA fragmentation and toxicity were not correlated with carcinogenicity. A significant correlation was found between DNA fragmentation and toxicity (r = 0.539). No correlation was found between DNA fragmentation and mutagenicity. The lack of correlation between DNA fragmentation and carcinogenicity is in contrast with previous results obtained with a family of hydrazine derivatives (12) and a group of nitrosocompounds (22). For these two groups of chemicals correlation between DNA fragmentation and carcinogenicity existed, but not between carcinogenicity and mutagenicity in the Ames' test. It is suggested that short term tests can perform very differently for different classes of chemicals.

  19. New Trends in Quantitative Assessment of the Corneal Barrier Function

    Directory of Open Access Journals (Sweden)

    Anton Guimerà

    2014-05-01

    Full Text Available The cornea is a very particular tissue due to its transparency and its barrier function as it has to resist against the daily insults of the external environment. In addition, maintenance of this barrier function is of crucial importance to ensure a correct corneal homeostasis. Here, the corneal epithelial permeability has been assessed in vivo by means of non-invasive tetrapolar impedance measurements, taking advantage of the huge impact of the ion fluxes in the passive electrical properties of living tissues. This has been possible by using a flexible sensor based in SU-8 photoresist. In this work, a further analysis focused on the validation of the presented sensor is performed by monitoring the healing process of corneas that were previously wounded. The obtained impedance measurements have been compared with the damaged area observed in corneal fluorescein staining images. The successful results confirm the feasibility of this novel method, as it represents a more sensitive in vivo and non-invasive test to assess low alterations of the epithelial permeability. Then, it could be used as an excellent complement to the fluorescein staining image evaluation.

  20. In vivo function of Pgβglu-1 in the release of acetophenones in white spruce

    Directory of Open Access Journals (Sweden)

    Melissa H. Mageroy

    2017-07-01

    Full Text Available Eastern spruce budworm (Choristoneura fumiferiana Clemens (ESBW is a major forest pest which feeds on young shoots of white spruce (Picea glauca and can cause landscape level economic and ecological losses. Release of acetophenone metabolites, piceol and pungenol, from their corresponding glycosides, picein and pungenin, can confer natural resistance of spruce to ESBW. A beta-glucosidase gene, Pgβglu-1, was recently discovered and the encoded enzyme was characterized in vitro to function in the release of the defensive acetophenone aglycons. Here we describe overexpression of Pgβglu-1 in a white spruce genotype whose metabolome contains the glucosylated acetophenones, but no detectable amounts of the aglycons. Transgenic overexpression of Pgβglu-1 resulted in release of the acetophenone aglycons in planta. This work provides in vivo evidence for the function of Pgβglu-1.

  1. In Vivo Determination of Vitamin D Function Using Transgenic Mice Carrying a Human Osteocalcin Luciferase Reporter Gene

    Directory of Open Access Journals (Sweden)

    Tomoko Nakanishi

    2013-01-01

    Full Text Available Vitamin D is an essential factor for ossification, and its deficiency causes rickets. Osteocalcin, which is a noncollagenous protein found in bone matrix and involved in mineralization and calcium ion homeostasis, is one of the major bone morphogenetic markers and is used in the evaluation of osteoblast maturation and osteogenic activation. We established transgenic mouse line expressing luciferase under the control of a 10-kb osteocalcin enhancer/promoter sequence. Using these transgenic mice, we evaluated the active forms of vitamins D2 and D3 for their bone morphogenetic function by in vivo bioluminescence. As the result, strong activity for ossification was observed with 1α,25-hydroxyvitamin D3. Our mouse system can offer a feasible detection method for assessment of osteogenic activity in the development of functional foods and medicines by noninvasive screening.

  2. In Vitro and In Vivo Evaluation of a Novel Ferrocyanide Functionalized Nanopourous Silica Decorporation Agent for Cesium in Rats

    Energy Technology Data Exchange (ETDEWEB)

    Timchalk, Charles; Creim, Jeffrey A.; Sukwarotwat, Vichaya; Wiacek, Robert J.; Addleman, Raymond S.; Fryxell, Glen E.; Yantasee, Wassana

    2010-09-01

    Novel decorporation agents are being developed to protect against radiological terrorist attacks. These sorbents, known as the self-assembled monolayer on mesoporous supports (SAMMS™), are hybrid materials where differing organic moieties are grafted onto mesoporous silica (SiO2). In vitro experiments focused on the evaluation, and optimization of SAMMS for capturing radiocesium (137Cs); based on these studies, a ferrocyanide copper (FC-Cu-EDA)-SAMMS was advanced for in vivo evaluation. In vivo experiments were conducted comparing the performance of the SAMMS vs. insoluble Prussian blue. Groups of jugular cannulated rats (4/treatment) were evaluated. Group I was administered 137Cs (~40 μgeq/kg) by intravenous (iv) injection and oral gavage; Group II was administered pre-bound 137Cs-SAMMS and sequential 137Cs + SAMMS (~61 ngeq/kg) by oral gavage; and Group III evaluated orally administered 137Cs (~0.06 μgeq/kg) followed by 0.1 g of either SAMMS or Prussian blue. Following dosing the rats were maintained in metabolism cages for 72 hour and blood, urine and fecal samples were collected for 137Cs analysis (gamma counting). Rats were then humanely euthanized, and selected tissues analyzed. Orally administered 137Cs was rapidly and well absorbed (~100% relative to iv dose), and the pharmacokinetics (blood, urine, feces & tissues) were very comparable to the iv dose group. For both exposures the urine and feces accounted for 20 and 3% of the dose, respectively. The prebound 137Cs-SAMMS was retained primarily within the feces (72% of the dose), with ~1.4% detected in the urine, suggesting that the 137Cs remained tightly bound to SAMMS. SAMMS & Prussian blue both effectively captured available 137Cs in the gut with feces accounting for 80-88% of the administered dose, while less than 2% was detected in the urine. This study suggests that the functionalized SAMMS out performs Prussian blue in vitro at low pH, but demonstrates comparable in vivo sequestration efficacy at

  3. Diverse Effects of L-arginine on Cardiac Function of Rats Subjected to Myocardial Ischemia and Reperfusion in vivo

    Institute of Scientific and Technical Information of China (English)

    Xiaoliang WANG; Feng LIANG; Xiangying JIAO; Lei LIU; Xiaojie BAI; Meixia LI; Jianmin ZHI; Huirong LIU

    2007-01-01

    In vivo administration of L-arginine at different time points during the course of myocardial ischemia and reperfusion (MI/R) has been shown to differentially regulate postischemic apoptosis.Cardiac function is one of the most important indexes used to judge the degree of myocardial injury.The present study attempted to determine whether in vivo administration of L-arginine at different stages of MI/R has a diverse influence on cardiac function of ischemic reperfused hearts and,if So,to investigate the mechanisms involved.Male adult rats were subjected to 30 min myocardial ischemia followed by 5 h reperfusion.An intravenous L-arginine bolus was given either 10 min before and 50 min after reperfusion (early treatment) or 3 h and 4 h after reperfusion (late treatment).Early treatment with L-arginine markedly increased the left ventricular systolic pressure (LVSP) and dP/dtmax,and decreased myocardial nitrotyrosine content.In strict contrast,late treatment with L-arginine resulted in a significant decrease in LVSP and dP/dtmx from 4 h to 5h after reperfusion,and increase in toxic peroxynitrite formation as measured by nitrotyrosine.These results suggest that the administration of L-arginine at different time points during the course of MI/R leads to diverse effects on cardiac dysfunction.Early supplementation decreased the nitrative stress and improved left ventricular function.However,late treatment with L-arginine increased the formation of peroxynitrite and aggravated cardiac functional injury.

  4. Genome-wide screens for in vivo Tinman binding sites identify cardiac enhancers with diverse functional architectures.

    Directory of Open Access Journals (Sweden)

    Hong Jin

    Full Text Available The NK homeodomain factor Tinman is a crucial regulator of early mesoderm patterning and, together with the GATA factor Pannier and the Dorsocross T-box factors, serves as one of the key cardiogenic factors during specification and differentiation of heart cells. Although the basic framework of regulatory interactions driving heart development has been worked out, only about a dozen genes involved in heart development have been designated as direct Tinman target genes to date, and detailed information about the functional architectures of their cardiac enhancers is lacking. We have used immunoprecipitation of chromatin (ChIP from embryos at two different stages of early cardiogenesis to obtain a global overview of the sequences bound by Tinman in vivo and their linked genes. Our data from the analysis of ~50 sequences with high Tinman occupancy show that the majority of such sequences act as enhancers in various mesodermal tissues in which Tinman is active. All of the dorsal mesodermal and cardiac enhancers, but not some of the others, require tinman function. The cardiac enhancers feature diverse arrangements of binding motifs for Tinman, Pannier, and Dorsocross. By employing these cardiac and non-cardiac enhancers in machine learning approaches, we identify a novel motif, termed CEE, as a classifier for cardiac enhancers. In vivo assays for the requirement of the binding motifs of Tinman, Pannier, and Dorsocross, as well as the CEE motifs in a set of cardiac enhancers, show that the Tinman sites are essential in all but one of the tested enhancers; although on occasion they can be functionally redundant with Dorsocross sites. The enhancers differ widely with respect to their requirement for Pannier, Dorsocross, and CEE sites, which we ascribe to their different position in the regulatory circuitry, their distinct temporal and spatial activities during cardiogenesis, and functional redundancies among different factor binding sites.

  5. In vivo readout of CFTR function: ratiometric measurement of CFTR-dependent secretion by individual, identifiable human sweat glands.

    Directory of Open Access Journals (Sweden)

    Jeffrey J Wine

    Full Text Available To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (~50 individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat was stimulated with a β-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ~0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with 'CFTR-related' conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.

  6. Genome-wide screens for in vivo Tinman binding sites identify cardiac enhancers with diverse functional architectures.

    Science.gov (United States)

    Jin, Hong; Stojnic, Robert; Adryan, Boris; Ozdemir, Anil; Stathopoulos, Angelike; Frasch, Manfred

    2013-01-01

    The NK homeodomain factor Tinman is a crucial regulator of early mesoderm patterning and, together with the GATA factor Pannier and the Dorsocross T-box factors, serves as one of the key cardiogenic factors during specification and differentiation of heart cells. Although the basic framework of regulatory interactions driving heart development has been worked out, only about a dozen genes involved in heart development have been designated as direct Tinman target genes to date, and detailed information about the functional architectures of their cardiac enhancers is lacking. We have used immunoprecipitation of chromatin (ChIP) from embryos at two different stages of early cardiogenesis to obtain a global overview of the sequences bound by Tinman in vivo and their linked genes. Our data from the analysis of ~50 sequences with high Tinman occupancy show that the majority of such sequences act as enhancers in various mesodermal tissues in which Tinman is active. All of the dorsal mesodermal and cardiac enhancers, but not some of the others, require tinman function. The cardiac enhancers feature diverse arrangements of binding motifs for Tinman, Pannier, and Dorsocross. By employing these cardiac and non-cardiac enhancers in machine learning approaches, we identify a novel motif, termed CEE, as a classifier for cardiac enhancers. In vivo assays for the requirement of the binding motifs of Tinman, Pannier, and Dorsocross, as well as the CEE motifs in a set of cardiac enhancers, show that the Tinman sites are essential in all but one of the tested enhancers; although on occasion they can be functionally redundant with Dorsocross sites. The enhancers differ widely with respect to their requirement for Pannier, Dorsocross, and CEE sites, which we ascribe to their different position in the regulatory circuitry, their distinct temporal and spatial activities during cardiogenesis, and functional redundancies among different factor binding sites.

  7. Circulating angiogenic cell function is inhibited by cortisol in vitro and associated with psychological stress and cortisol in vivo.

    Science.gov (United States)

    Aschbacher, Kirstin; Derakhshandeh, Ronak; Flores, Abdiel J; Narayan, Shilpa; Mendes, Wendy Berry; Springer, Matthew L

    2016-05-01

    Psychological stress and glucocorticoids are associated with heightened cardiovascular disease risk. We investigated whether stress or cortisol would be associated with reduced circulating angiogenic cell (CAC) function, an index of impaired vascular repair. We hypothesized that minority-race individuals who experience threat in interracial interactions would exhibit reduced CAC function, and that this link might be explained by cortisol. To test this experimentally, we recruited 106 African American participants for a laboratory interracial interaction task, in which they received socially evaluative feedback from Caucasian confederates. On a separate day, a subset of 32 participants (mean age=26years, 47% female) enrolled in a separate biological substudy and provided blood samples for CAC isolation and salivary samples to quantify the morning peak in cortisol (the cortisol awakening response, CAR). CAC function was quantified using cell culture assays of migration to vascular endothelial growth factor (VEGF) and secretion of VEGF into the culture medium. Heightened threat in response to an interracial interaction and trait anxiety in vivo were both associated with poorer CAC migratory function in vitro. Further, threat and poorer sustained attention during the interracial interaction were associated with a higher CAR, which in turn, was related to lower CAC sensitivity to glucocorticoids. In vitro, higher doses of cortisol impaired CAC migratory function and VEGF protein secretion. The glucocorticoid receptor antagonist RU486 reversed this functional impairment. These data identify a novel, neuroendocrine pathway by which psychological stress may reduce CAC function, with potential implications for cardiovascular health.

  8. In vivo visuotopic brain mapping with manganese-enhanced MRI and resting-state functional connectivity MRI.

    Science.gov (United States)

    Chan, Kevin C; Fan, Shu-Juan; Chan, Russell W; Cheng, Joe S; Zhou, Iris Y; Wu, Ed X

    2014-04-15

    The rodents are an increasingly important model for understanding the mechanisms of development, plasticity, functional specialization and disease in the visual system. However, limited tools have been available for assessing the structural and functional connectivity of the visual brain network globally, in vivo and longitudinally. There are also ongoing debates on whether functional brain connectivity directly reflects structural brain connectivity. In this study, we explored the feasibility of manganese-enhanced MRI (MEMRI) via 3 different routes of Mn(2+) administration for visuotopic brain mapping and understanding of physiological transport in normal and visually deprived adult rats. In addition, resting-state functional connectivity MRI (RSfcMRI) was performed to evaluate the intrinsic functional network and structural-functional relationships in the corresponding anatomical visual brain connections traced by MEMRI. Upon intravitreal, subcortical, and intracortical Mn(2+) injection, different topographic and layer-specific Mn enhancement patterns could be revealed in the visual cortex and subcortical visual nuclei along retinal, callosal, cortico-subcortical, transsynaptic and intracortical horizontal connections. Loss of visual input upon monocular enucleation to adult rats appeared to reduce interhemispheric polysynaptic Mn(2+) transfer but not intra- or inter-hemispheric monosynaptic Mn(2+) transport after Mn(2+) injection into visual cortex. In normal adults, both structural and functional connectivity by MEMRI and RSfcMRI was stronger interhemispherically between bilateral primary/secondary visual cortex (V1/V2) transition zones (TZ) than between V1/V2 TZ and other cortical nuclei. Intrahemispherically, structural and functional connectivity was stronger between visual cortex and subcortical visual nuclei than between visual cortex and other subcortical nuclei. The current results demonstrated the sensitivity of MEMRI and RSfcMRI for assessing the

  9. Structure-function studies of STAR family Quaking proteins bound to their in vivo RNA target sites

    Energy Technology Data Exchange (ETDEWEB)

    Teplova, Marianna; Hafner, Markus; Teplov, Dmitri; Essig, Katharina; Tuschl, Thomas; Patel, Dinshaw J. [MSKCC; (Rockefeller)

    2013-09-27

    Mammalian Quaking (QKI) and its Caenorhabditis elegans homolog, GLD-1 (defective in germ line development), are evolutionarily conserved RNA-binding proteins, which post-transcriptionally regulate target genes essential for developmental processes and myelination. We present X-ray structures of the STAR (signal transduction and activation of RNA) domain, composed of Qua1, K homology (KH), and Qua2 motifs of QKI and GLD-1 bound to high-affinity in vivo RNA targets containing YUAAY RNA recognition elements (RREs). The KH and Qua2 motifs of the STAR domain synergize to specifically interact with bases and sugar-phosphate backbones of the bound RRE. Qua1-mediated homodimerization generates a scaffold that enables concurrent recognition of two RREs, thereby plausibly targeting tandem RREs present in many QKI-targeted transcripts. Structure-guided mutations reduced QKI RNA-binding affinity in vitro and in vivo, and expression of QKI mutants in human embryonic kidney cells (HEK293) significantly decreased the abundance of QKI target mRNAs. Overall, our studies define principles underlying RNA target selection by STAR homodimers and provide insights into the post-transcriptional regulatory function of mammalian QKI proteins.

  10. Identification of the in vivo function of the high-efficiency D-mannonate dehydratase in Caulobacter crescentus NA1000 from the enolase superfamily.

    Science.gov (United States)

    Wichelecki, Daniel J; Graff, Dylan C; Al-Obaidi, Nawar; Almo, Steven C; Gerlt, John A

    2014-07-01

    The d-mannonate dehydratase (ManD) subgroup of the enolase superfamily contains members with varying catalytic activities (high-efficiency, low-efficiency, or no activity) that dehydrate d-mannonate and/or d-gluconate to 2-keto-3-deoxy-d-gluconate [Wichelecki, D. J., et al. (2014) Biochemistry 53, 2722-2731]. Despite extensive in vitro characterization, the in vivo physiological role of a ManD has yet to be established. In this study, we report the in vivo functional characterization of a high-efficiency ManD from Caulobacter crescentus NA1000 (UniProt entry B8GZZ7) by in vivo discovery of its essential role in d-glucuronate metabolism. This in vivo functional annotation may be extended to ~50 additional proteins.

  11. Functional Analysis of Vascularized Collagen/Fibrin Templates by MRI In Vivo

    NARCIS (Netherlands)

    Sun, W.; Sun, Y; Klar, A.S.; Geutjes, P.; Reichmann, E.; Heerschap, A.; Oosterwijk, E.

    2016-01-01

    Functional monitoring of the fate of implanted templates, which restore the function of lost tissues, is still a challenge. Whereas histology can give excellent insight into material and tissue remodeling, longitudinal studies are hampered by the invasive character. Noninvasive imaging techniques,

  12. In vivo evidence of functional and anatomical stripe-based subdivisions in human V2 and V3.

    Science.gov (United States)

    Dumoulin, Serge O; Harvey, Ben M; Fracasso, Alessio; Zuiderbaan, Wietske; Luijten, Peter R; Wandell, Brian A; Petridou, Natalia

    2017-04-07

    Visual cortex contains a hierarchy of visual areas. The earliest cortical area (V1) contains neurons responding to colour, form and motion. Later areas specialize on processing of specific features. The second visual area (V2) in non-human primates contains a stripe-based anatomical organization, initially defined using cytochrome-oxidase staining of post-mortem tissue. Neurons in these stripes have been proposed to serve distinct functional specializations, e.g. processing of color, form and motion. These stripes represent an intermediate stage in visual hierarchy and serve a key role in the increasing functional specialization of visual areas. Using sub-millimeter high-field functional and anatomical MRI (7T), we provide in vivo evidence for stripe-based subdivisions in humans. Using functional MRI, we contrasted responses elicited by stimuli alternating at slow and fast temporal frequencies. We revealed stripe-based subdivisions in V2 ending at the V1/V2 border. The human stripes reach into V3. Using anatomical MRI optimized for myelin contrast within gray matter, we also observe a stripe pattern. Stripe subdivisions preferentially responding to fast temporal frequencies are more myelinated. As such, functional and anatomical measures provide independent and converging evidence for functional organization into striped-based subdivisions in human V2 and V3.

  13. Melanopsin Phototransduction Contributes to Light-Evoked Choroidal Expansion and Rod L-Type Calcium Channel Function In Vivo

    Science.gov (United States)

    Berkowitz, Bruce A.; Schmidt, Tiffany; Podolsky, Robert H.; Roberts, Robin

    2016-01-01

    Purpose In humans, rodents, and pigeons, the dark → light transition signals nonretinal brain tissue to increase choroidal thickness, a major control element of choroidal blood flow, and thus of photoreceptor and retinal pigment epithelium function. However, it is unclear which photopigments in the retina relay the light signal to the brain. Here, we test the hypothesis that melanopsin (Opn4)-regulated phototransduction modulates light-evoked choroidal thickness expansion in mice. Methods Two-month-old C57Bl/6 wild-type (B6), 4- to 5-month-old C57Bl/6/129S6 wild-type (B6 + S6), and 2-month-old melanopsin knockout (Opn4−/−) on a B6 + S6 background were studied. Retinal anatomy was evaluated in vivo by optical coherence tomography and MRI. Choroidal thickness in dark and light were measured by diffusion-weighted MRI. Rod cell L-type calcium channel (LTCC) function in dark and light (manganese-enhanced MRI [MEMRI]) was also measured. Results Opn4−/− mice did not show the light-evoked expansion of choroidal thickness observed in B6 and B6 + S6 controls. Additionally, Opn4−/− mice had lower than normal rod cell and inner retinal LTCC function in the dark but not in the light. These deficits were not due to structural abnormalities because retinal laminar architecture and thickness, and choroidal thickness in the Opn4−/− mice were similar to controls. Conclusions First time evidence is provided that melanopsin phototransduction contributes to dark → light control of murine choroidal thickness. The data also highlight a contribution in vivo of melanopsin phototransduction to rod cell and inner retinal depolarization in the dark. PMID:27727394

  14. In vivo evaluation of the inhibitory capacity of human plasma on exogenous surfactant function

    NARCIS (Netherlands)

    B.F. Lachmann (Burkhard); E.P. Eijking (Eric); K.L. So; D.A.M.P.J. Gommers (Diederik)

    1994-01-01

    textabstractObjective: The adult respiratory distress syndrome (ARDS) and neonatal respiratory distress syndrome (RDS) are characterized by high permeability pulmonary edema which contains plasma-derived proteins inhibiting pulmonary surfactant function. Currently, discussion continues as to what do

  15. Could quantitative liver function tests gain wide acceptance among hepatologists?

    Institute of Scientific and Technical Information of China (English)

    Giovanni Tarantino

    2009-01-01

    It has been emphasized that the assessment of residual liver function is of paramount importance to determine the following: severity of acute or chronic liver diseases independent of etiology; long-term prognosis; step-bystep disease progression; surgical risk; and efficacy of antiviral treatment. The most frequently used tools are the galactose elimination capacity to asses hepatocyte cytosol activity, plasma clearance of indocyanine green to assess excretory function, and antipyrine clearance to estimate microsomal activity. However, a widely accepted liver test (not necessarily a laboratory one) to assess quantitative functional hepatic reserve still needs to be established, although there have been various proposals. Furthermore, who are the operators that should order these tests? Advances in analytic methods are expected to allow quantitative liver function tests to be used in clinical practice.

  16. Characterization of the RND family of multidrug efflux pumps: in silico to in vivo confirmation of four functionally distinct subgroups.

    Science.gov (United States)

    Godoy, Patricia; Molina-Henares, Antonio J; de la Torre, Jesús; Duque, Estrella; Ramos, Juan L

    2010-11-01

    We have developed a generalized profile that identifies members of the root-nodulation-cell-division (RND) family of efflux pumps and classifies them into four functional subfamilies. According to Z-score values, efflux pumps can be grouped by their metabolic function, thus making it possible to distinguish pumps involved in antibiotic resistance (group 1) from those involved in metal resistance (group 3). In silico data regarding efflux pumps in group 1 were validated after identification of RND efflux pumps in a number of environmental microbes that were isolated as resistant to ethidium bromide. Analysis of the Pseudomonas putida KT2440 genome identified efflux pumps in all groups. A collection of mutants in efflux pumps and a screening platform consisting of 50 drugs were created to assign a function to the efflux pumps. We validated in silico data regarding efflux pumps in groups 1 and 3 using 9 different mutants. Four mutants belonging to group 2 were found to be more sensitive than the wild-type to oxidative stress-inducing agents such as bipyridyl and methyl viologen. The two remaining mutants belonging to group 4 were found to be more sensitive than the parental to tetracycline and one of them was particularly sensitive to rubidium and chromate. By effectively combining in vivo data with generalized profiles and gene annotation data, this approach allowed the assignment, according to metabolic function, of both known and uncharacterized RND efflux pumps into subgroups, thereby providing important new insight into the functions of proteins within this family.

  17. Facile Peptides Functionalization of Lanthanide-Based Nanocrystals through Phosphorylation Tethering for Efficient in Vivo NIR-to-NIR Bioimaging.

    Science.gov (United States)

    Yao, Chi; Wang, Peiyuan; Wang, Rui; Zhou, Lei; El-Toni, Ahmed Mohamed; Lu, Yiqing; Li, Xiaomin; Zhang, Fan

    2016-02-02

    Peptide modification of nanoparticles is a challenging task for bioapplications. Here, we show that noncovalent surface engineering based on ligand exchange of peptides for lanthanide based upconversion and downconversion near-infrared (NIR) luminescent nanoparticles can be efficiently realized by modifying the hydroxyl functional group of a side grafted serine of peptides into a phosphate group (phosphorylation). By using the phosphorylated peptide with the arginine-glycine-aspartic acid (RGD) targeting motifs as typical examples, the modification allows improving the selectivity, sensitivity, and signal-to-noise ratio for the cancer targeting and bioimaging and reducing the toxicity derived from nonspecific interactions of nanoparticles with cells. The in vivo NIR bioimaging signal could even be detected at low injection amounts down to 20 μg per animal.

  18. The Angiotensin-(1-7)/Mas Axis Improves Pancreatic β-Cell Function in Vitro and in Vivo.

    Science.gov (United States)

    Sahr, Anika; Wolke, Carmen; Maczewsky, Jonas; Krippeit-Drews, Peter; Tetzner, Anja; Drews, Gisela; Venz, Simone; Gürtler, Sarah; van den Brandt, Jens; Berg, Sabine; Döring, Paula; Dombrowski, Frank; Walther, Thomas; Lendeckel, Uwe

    2016-12-01

    The angiotensin-converting enzyme 2/angiotensin (Ang)-(1-7)/Mas axis of the renin-angiotensin system often opposes the detrimental effects of the angiotensin-converting enzyme/Ang II/Ang II type 1 receptor axis and has been associated with beneficial effects on glucose homeostasis, whereas underlying mechanisms are mostly unknown. Here we investigate the effects of Ang-(1-7) and its receptor Mas on β-cell function. Isolated islets from Mas-deficient and wild-type mice were stimulated with Ang-(1-7) or its antagonists and effects on insulin secretion determined. Islets' cytoplasmic calcium and cAMP concentrations, mRNA amounts of Ins1, Ins2, Pdx1, and Mafa and effects of inhibitors of cAMP downstream signaling were determined. Ang-(1-7) was also applied to mice by osmotic pumps for 14 days and effects on glucose tolerance and insulin secretion were assessed. Ang-(1-7) increased insulin secretion from wild-type islets, whereas antagonists and genetic Mas deficiency led to reduced insulin secretion. The Mas-dependent effects of Ang-(1-7) on insulin secretion did not result from changes in insulin gene expression or changes in the excitation-secretion coupling but from increased intracellular cAMP involving exchange protein activated directly by cAMP. Administration of Ang-(1-7) in vivo had only marginal effects on glucose tolerance in wild-type mice but still resulted in improved insulin secretion from islets isolated of these mice. Interestingly, although less pronounced than in wild types, Ang-(1-7) still affected insulin secretion in Mas-deficient islets. The data indicate a significant function of Ang-(1-7) in the regulation of insulin secretion from mouse islets in vitro and in vivo, mainly, but not exclusively, by Mas-dependent signaling, modulating the accessory pathway of insulin secretion via increase in cAMP.

  19. Renal effects of nabumetone, a COX-2 antagonist: impairment of function in isolated perfused rat kidneys contrasts with preserved renal function in vivo.

    Science.gov (United States)

    Reichman, J; Cohen, S; Goldfarb, M; Shina, A; Rosen, S; Brezis, M; Karmeli, F; Heyman, S N

    2001-01-01

    The constitutive cyclooxygenase (COX)-1 enzyme has been considered the physiologically important isoform for prostaglandin synthesis in the normal kidney. It has, therefore, been suggested that selective inhibitors of the 'inducible' isoform (COX-2) may be free from renal adverse effects. We studied the renal effects of the predominantly COX-2 antagonist nabumetone in isolated perfused kidneys. As compared with controls, kidneys removed after in vivo administration of oral nabumetone (15 mg/kg) disclosed altered renal function with reduced glomerular filtration rate, filtration fraction, and urine volume and enhanced hypoxic outer medullary tubular damage. By contrast, renal function and morphology were not affected in vivo by nabumetone or its active metabolite 6-methoxy-2-naphthylacetic acid. The latter agent (10-20 mg/kg i.v.) did not significantly alter renal microcirculation, as opposed to a selective substantial reduction in medullary blood flow noted with the nonselective COX inhibitor indomethacin (5 mg/kg i.v.). In a rat model of acute renal failure, induced by concomitant administration of radiocontrast, nitric oxide synthase, and COX inhibitors, the decline in kidney function and the extent of hypoxic medullary damage with oral nabumetone (80 mg/kg) were comparable to a control group, and significantly less than those induced by indomethacin. In rats subjected to daily oral nabumetone for 3 consecutive weeks, renal function and morphology were preserved as well. Both nabumetone and 6-methoxy-2-naphthylacetic acid reduced renal parenchymal prostaglandin E2 to the same extent as indomethacin. It is concluded that while nabumetone adversely affects renal function and may intensify hypoxic medullary damage ex vivo, rat kidneys are not affected by this agent in vivo, both in acute and chronic studies. COX selectivity may not explain the renal safety of nabumetone.

  20. Cine-MRI versus two-dimensional echocardiography to measure in vivo left ventricular function in rat heart.

    Science.gov (United States)

    Stuckey, Daniel J; Carr, Carolyn A; Tyler, Damian J; Clarke, Kieran

    2008-08-01

    Two-dimensional echocardiography is the most commonly used non-invasive method for measuring in vivo cardiac function in experimental animals. In humans, measurements of cardiac function made using cine-MRI compare favourably with those made using echocardiography. However, no rigorous comparison has been made in small animals. Here, standard short-axis two-dimensional (2D) echocardiography (2D-echo) and cine-MRI measurements were made in the same rats, both control and after chronic myocardial infarction. Correlations between the two techniques were found for end diastolic area, stroke area and ejection fraction, but cine-MRI measurements of ejection fraction were 12+/-6% higher than those made using 2D-echo, because of the 1.8-fold higher temporal resolution of the MRI technique (4.6 ms vs 8.3 ms). Repeated measurements on the same group of rats over several days showed that the cine-MRI technique was more reproducible than 2D-echo, in that 2D-echo would require five times more animals to find a statistically significant difference. In summary, caution should be exercised when comparing functional results acquired using short-axis 2D-echo vs cine-MRI. The accuracy of cine-MRI allows identification of alterations in heart function that may be missed when using 2D-echo.

  1. Physarum polycephalum for Studying the Function of Histone Modifications In Vivo.

    Science.gov (United States)

    Menil-Philippot, Vanessa; Thiriet, Christophe

    2017-01-01

    Histone modifications have been widely correlated with genetic activities. However, how these posttranslational modifications affect the dynamics and the structure of chromatin is poorly understood. Here, we describe the incorporation of the exogenous histone proteins into the slime mold Physarum polycephalum, which has been revealed to be a valuable tool for examining different facets of the function histones in chromatin dynamics like replication-coupled chromatin assembly, histone exchange, and nucleosome turnover.

  2. Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo.

    Directory of Open Access Journals (Sweden)

    Kristina Preuße

    2015-06-01

    Full Text Available Notch signalling is a fundamental pathway that shapes the developing embryo and sustains adult tissues by direct communication between ligand and receptor molecules on adjacent cells. Among the ligands are two Delta paralogues, DLL1 and DLL4, that are conserved in mammals and share a similar structure and sequence. They activate the Notch receptor partly in overlapping expression domains where they fulfil redundant functions in some processes (e.g. maintenance of the crypt cell progenitor pool. In other processes, however, they appear to act differently (e.g. maintenance of foetal arterial identity raising the questions of how similar DLL1 and DLL4 really are and which mechanism causes the apparent context-dependent divergence. By analysing mice that conditionally overexpress DLL1 or DLL4 from the same genomic locus (Hprt and mice that express DLL4 instead of DLL1 from the endogenous Dll1 locus (Dll1Dll4ki, we found functional differences that are tissue-specific: while DLL1 and DLL4 act redundantly during the maintenance of retinal progenitors, their function varies in the presomitic mesoderm (PSM where somites form in a Notch-dependent process. In the anterior PSM, every cell expresses both Notch receptors and ligands, and DLL1 is the only activator of Notch while DLL4 is not endogenously expressed. Transgenic DLL4 cannot replace DLL1 during somitogenesis and in heterozygous Dll1Dll4ki/+ mice, the Dll1Dll4ki allele causes a dominant segmentation phenotype. Testing several aspects of the complex Notch signalling system in vitro, we found that both ligands have a similar trans-activation potential but that only DLL4 is an efficient cis-inhibitor of Notch signalling, causing a reduced net activation of Notch. These differential cis-inhibitory properties are likely to contribute to the functional divergence of DLL1 and DLL4.

  3. Noninvasive assessment of gene transfer and expression by in vivo functional and morphologic imaging in a rabbit tumor model.

    Directory of Open Access Journals (Sweden)

    Murali K Ravoori

    animal tumor model. Using clinical machines, morphologic imaging contributes to functional imaging for quantifying SSTR2-based reporter expression in vivo.

  4. Enhancement of Extracts from Celastrus orbiculatus on Maturation and Function of Dendritic Cells in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    QIAN Ya-yun; ZHANG Hua; YUAN Lin; HOU Ying; LIU Wei-wei; LIU Yan-qing

    2010-01-01

    Objective To examine the immunoregulation of Celastrus orbiculatus extracts(COE),a traditional Chinese medicine,on maturation and function of dendritic cells(DCs)in vitro and in vivo.Methods In vitro,after treated with COE indifferent nontoxic concentrations(0,10,20,40,80,and 160 μg/mL)for 5 d,the surface immunological molecules andcytokine secretion of mice bone marrow-derived DCs in response to COE were analyzed by flow cytometric analysis(FACS)and enzyme linked immunosorbent assay(ELISA),respectively.In vivo,mouse hepatoma cells(Hepal-6,1 ×106)were injected sc and were treated with different dosages of COE(10,20 or 40 mg/kg/d).Effects on tumor growth were determined by tumor volume and histology analysis after 28 d administration of COE.The relative proportions ofmature DCs and CD8+ T cells were measured in mononuclear cells that had been isolated from spleen by FACS.Results COE stimulated IL-2 and IFN-γ secretion of DCs,simultaneously enhanced the maturation of DCs byenhancing immunological molecule(CD40,CDS0,CD86,H-2Kb,and I-Ab)expression in a dose-dependent manner.Furthermore,the chcmotactic responses of DCs were significantly higher in COE-treated than untreated DCs,in association with higher chcmokine receptor 7 expression.Furthermore,COE increased DCs produce IFN-γ and IL-2 ina dose-dependent manner when the concentration of COE less than 40 μg/mL,decreased DCs produce IL-10 and IL-4also in a dose-dependent manner.In in vivo studies,COE can not only suppress growth of malignant hepatocellularcarcinomas but also stimulate maturation of DCs,associated with strongly enhanced CD8+ CTL responses.ConclusionThese data provide new insight into the mechanism of action of COE and indicate that the stimulation of maturation andfunction of DCs by COE contributes to its immunoregulatory effects.

  5. In Vivo Shoulder Function After Surgical Repair of a Torn Rotator Cuff

    Science.gov (United States)

    Bey, Michael J.; Peltz, Cathryn D.; Ciarelli, Kristin; Kline, Stephanie K.; Divine, George W.; van Holsbeeck, Marnix; Muh, Stephanie; Kolowich, Patricia A.; Lock, Terrence R.; Moutzouros, Vasilios

    2015-01-01

    Background Surgical repair of a torn rotator cuff is based on the belief that repairing the tear is necessary to restore normal glenohumeral joint (GHJ) mechanics and achieve a satisfactory clinical outcome. Hypothesis Dynamic joint function is not completely restored by rotator cuff repair, thus compromising shoulder function and potentially leading to long-term disability. Study Design Controlled laboratory study and Case series; Level of evidence, 4. Methods Twenty-one rotator cuff patients and 35 control participants enrolled in the study. Biplane radiographic images were acquired bilaterally from each patient during coronal-plane abduction. Rotator cuff patients were tested at 3, 12, and 24 months after repair of a supraspinatus tendon tear. Control participants were tested once. Glenohumeral joint kinematics and joint contact patterns were accurately determined from the biplane radiographic images. Isometric shoulder strength and patient-reported outcomes were measured at each time point. Ultrasound imaging assessed rotator cuff integrity at 24 months after surgery. Results Twenty of 21 rotator cuff repairs appeared intact at 24 months after surgery. The humerus of the patients’ repaired shoulder was positioned more superiorly on the glenoid than both the patients’ contralateral shoulder and the dominant shoulder of control participants. Patient-reported outcomes improved significantly over time. Shoulder strength also increased over time, although strength deficits persisted at 24 months for most patients. Changes over time in GHJ mechanics were not detected for either the rotator cuff patients’ repaired or contralateral shoulders. Clinical outcome was associated with shoulder strength but not GHJ mechanics. Conclusion Surgical repair of an isolated supraspinatus tear may be sufficient to keep the torn rotator cuff intact and achieve satisfactory patient-reported outcomes, but GHJ mechanics and shoulder strength are not fully restored with current

  6. The Relationship between Dyslipidemia and Acute Axonal Function in Type 2 Diabetes Mellitus In Vivo.

    Science.gov (United States)

    Kwai, Natalie C G; Nigole, William; Poynten, Ann M; Brown, Christopher; Krishnan, Arun V

    2016-01-01

    Diabetic peripheral neuropathy (DPN) is a common and debilitating complication of diabetes mellitus. Treatment largely consists of symptom alleviation and there is a need to identify therapeutic targets for prevention and treatment of DPN. The objective of this study was to utilise novel neurophysiological techniques to investigate axonal function in patients with type 2 diabetes and to prospectively determine their relationship to serum lipids in type 2 diabetic patients. Seventy-one patients with type 2 diabetes were consecutively recruited and tested. All patients underwent thorough clinical neurological assessments including nerve conduction studies, and median motor axonal excitability studies. Studies were also undertaken in age matched normal control subjects(n = 42). Biochemical studies, including serum lipid levels were obtained in all patients. Patient excitability data was compared to control data and linear regression analysis was performed to determine the relationship between serum triglycerides and low density lipoproteins and excitability parameters typically abnormal in type 2 diabetic patients. Patient mean age was 64.2±2.3 years, mean glycosylated haemoglobin (HbA1c%) was 7.8±0.3%, mean triglyceride concentration was 1.6±0.1 mmol/L and mean cholesterol concentration was 4.1±0.2mmol/L. Compared to age matched controls, median motor axonal excitability studies indicated axonal dysfunction in type 2 diabetic patients as a whole (T2DM) and in a subgroup of the patients without DPN (T2DM-NN). These included reduced percentage threshold change during threshold electrotonus at 10-20ms depolarising currents (TEd10-20ms)(controls 68.4±0.8, T2DM63.9±0.8, T2DM-NN64.8±1.6%,Plipoprotein concentration when adjusted for renal function, a separate risk factor for neuropathy development. Our findings indicate that acutely, serum lipids do not exert an acute effect on axonal function in type 2 diabetic patients: TEd(10-20ms)(1.2(-1.4,3.8);P = 0.4) and

  7. High sensitivity contrast enhanced optical coherence tomography for functional in vivo imaging

    Science.gov (United States)

    Liba, Orly; SoRelle, Elliott D.; Sen, Debasish; de la Zerda, Adam

    2017-02-01

    In this study, we developed and applied highly-scattering large gold nanorods (LGNRs) and custom spectral detection algorithms for high sensitivity contrast-enhanced optical coherence tomography (OCT). We were able to detect LGNRs at a concentration as low as 50 pM in blood. We used this approach for noninvasive 3D imaging of blood vessels deep in solid tumors in living mice. Additionally, we demonstrated multiplexed imaging of spectrally-distinct LGNRs that enabled observations of functional drainage in lymphatic networks. This method, which we call MOZART, provides a platform for molecular imaging and characterization of tissue noninvasively at cellular resolution.

  8. Menstrual Blood-Derived Stem Cells: In Vitro and In Vivo Characterization of Functional Effects.

    Science.gov (United States)

    Rodrigues, Maria Carolina Oliveira; Lippert, Trenton; Nguyen, Hung; Kaelber, Sussannah; Sanberg, Paul R; Borlongan, Cesar V

    2016-01-01

    Accumulating evidence has demonstrated that menstrual blood stands as a viable source of stem cells. Menstrual blood-derived stem cells (MenSCs) are morphologically and functionally similar to cells directly extracted from the endometrium, and present dual expression of mesenchymal and embryonic cell markers, thus becoming interesting tools for regenerative medicine. Functional reports show higher proliferative and self-renewal capacities than bone marrow-derived stem cells, as well as successful differentiation into hepatocyte-like cells, glial-like cells, endometrial stroma-like cells, among others. Moreover, menstrual blood stem cells may be used with increased efficiency in reprogramming techniques for induced Pluripotent Stem cell (iPS) generation. Experimental studies have shown successful treatment of stroke, colitis, limb ischemia, coronary disease, Duchenne's muscular atrophy and streptozotocin-induced type 1 diabetes animal models with MenSCs. As we envision an off-the-shelf product for cell therapy, cryopreserved MenSCs appear as a feasible clinical product. Clinical applications, although still very limited, have great potential and ongoing studies should be disclosed in the near future.

  9. Ligand binding-dependent functions of the lipocalin NLaz: an in vivo study in Drosophila.

    Science.gov (United States)

    Ruiz, Mario; Ganfornina, Maria D; Correnti, Colin; Strong, Roland K; Sanchez, Diego

    2014-04-01

    Lipocalins are small extracellular proteins mostly described as lipid carriers. The Drosophila lipocalin NLaz (neural Lazarillo) modulates the IIS pathway and regulates longevity, stress resistance, and behavior. Here, we test whether a native hydrophobic pocket structure is required for NLaz to perform its functions. We use a point mutation altering the binding pocket (NLaz(L130R)) and control mutations outside NLaz binding pocket. Tryptophan fluorescence titration reveals that NLaz(L130R) loses its ability to bind ergosterol and the pheromone 7(z)-tricosene but retains retinoic acid binding. Using site-directed transgenesis in Drosophila, we test the functionality of the ligand binding-altered lipocalin at the organism level. NLaz-dependent life span reduction, oxidative stress and starvation sensitivity, aging markers accumulation, and deficient courtship are rescued by overexpression of NLaz(WT), but not of NLaz(L130R). Transcriptional responses to aging and oxidative stress show a large set of age-responsive genes dependent on the integrity of NLaz binding pocket. Inhibition of IIS activity and modulation of oxidative stress and infection-responsive genes are binding pocket-dependent processes. Control of energy metabolites on starvation appears to be, however, insensitive to the modification of the NLaz binding pocket.

  10. The Relationship between Dyslipidemia and Acute Axonal Function in Type 2 Diabetes Mellitus In Vivo

    Science.gov (United States)

    Kwai, Natalie C. G.; Nigole, William; Poynten, Ann M.; Brown, Christopher; Krishnan, Arun V.

    2016-01-01

    Objectives Diabetic peripheral neuropathy (DPN) is a common and debilitating complication of diabetes mellitus. Treatment largely consists of symptom alleviation and there is a need to identify therapeutic targets for prevention and treatment of DPN. The objective of this study was to utilise novel neurophysiological techniques to investigate axonal function in patients with type 2 diabetes and to prospectively determine their relationship to serum lipids in type 2 diabetic patients. Methods Seventy-one patients with type 2 diabetes were consecutively recruited and tested. All patients underwent thorough clinical neurological assessments including nerve conduction studies, and median motor axonal excitability studies. Studies were also undertaken in age matched normal control subjects(n = 42). Biochemical studies, including serum lipid levels were obtained in all patients. Patient excitability data was compared to control data and linear regression analysis was performed to determine the relationship between serum triglycerides and low density lipoproteins and excitability parameters typically abnormal in type 2 diabetic patients. Results Patient mean age was 64.2±2.3 years, mean glycosylated haemoglobin (HbA1c%) was 7.8±0.3%, mean triglyceride concentration was 1.6±0.1 mmol/L and mean cholesterol concentration was 4.1±0.2mmol/L. Compared to age matched controls, median motor axonal excitability studies indicated axonal dysfunction in type 2 diabetic patients as a whole (T2DM) and in a subgroup of the patients without DPN (T2DM-NN). These included reduced percentage threshold change during threshold electrotonus at 10–20ms depolarising currents (TEd10–20ms)(controls 68.4±0.8, T2DM63.9±0.8, T2DM-NN64.8±1.6%,P<0.05) and superexcitability during the recovery cycle (controls-22.5±0.9, T2DM-17.5±0.8, T2DM-NN-17.3±1.6%,P<0.05). Linear regression analysis revealed no associations between changes in axonal function and either serum triglyceride or low density

  11. Functional genomics analysis of vitamin D effects on CD4+ T cells in vivo in experimental autoimmune encephalomyelitis ‬

    KAUST Repository

    Zeitelhofer, Manuel

    2017-02-15

    Vitamin D exerts multiple immunomodulatory functions and has been implicated in the etiology and treatment of several autoimmune diseases, including multiple sclerosis (MS). We have previously reported that in juvenile/adolescent rats, vitamin D supplementation protects from experimental autoimmune encephalomyelitis (EAE), a model of MS. Here we demonstrate that this protective effect associates with decreased proliferation of CD4+ T cells and lower frequency of pathogenic T helper (Th) 17 cells. Using transcriptome, methylome, and pathway analyses in CD4+ T cells, we show that vitamin D affects multiple signaling and metabolic pathways critical for T-cell activation and differentiation into Th1 and Th17 subsets in vivo. Namely, Jak/Stat, Erk/Mapk, and Pi3K/Akt/mTor signaling pathway genes were down-regulated upon vitamin D supplementation. The protective effect associated with epigenetic mechanisms, such as (i) changed levels of enzymes involved in establishment and maintenance of epigenetic marks, i.e., DNA methylation and histone modifications; (ii) genome-wide reduction of DNA methylation, and (iii) up-regulation of noncoding RNAs, including microRNAs, with concomitant down-regulation of their protein-coding target RNAs involved in T-cell activation and differentiation. We further demonstrate that treatment of myelin-specific T cells with vitamin D reduces frequency of Th1 and Th17 cells, down-regulates genes in key signaling pathways and epigenetic machinery, and impairs their ability to transfer EAE. Finally, orthologs of nearly 50% of candidate MS risk genes and 40% of signature genes of myelin-reactive T cells in MS changed their expression in vivo in EAE upon supplementation, supporting the hypothesis that vitamin D may modulate risk for developing MS.

  12. Regulation of translation in haloarchaea: 5'- and 3'-UTRs are essential and have to functionally interact in vivo.

    Directory of Open Access Journals (Sweden)

    Mariam Brenneis

    Full Text Available Recently a first genome-wide analysis of translational regulation using prokaryotic species had been performed which revealed that regulation of translational efficiency plays an important role in haloarchaea. In fact, the fractions of genes under differential growth phase-dependent translational control in the two species Halobacterium salinarum and Haloferax volcanii were as high as in eukaryotes. However, nothing is known about the mechanisms of translational regulation in archaea. Therefore, two genes exhibiting opposing directions of regulation were selected to unravel the importance of untranslated regions (UTRs for differential translational control in vivo.Differential translational regulation in exponentially growing versus stationary phase cells was studied by comparing translational efficiencies using a reporter gene system. Translational regulation was not observed when 5'-UTRs or 3'-UTRs alone were fused to the reporter gene. However, their simultaneous presence was sufficient to transfer differential translational control from the native transcript to the reporter transcript. This was true for both directions of translational control. Translational regulation was completely abolished when stem loops in the 5'-UTR were changed by mutagenesis. An "UTR-swap" experiment demonstrated that the direction of translational regulation is encoded in the 3'-UTR, not in the 5'-UTR. While much is known about 5'-UTR-dependent translational control in bacteria, the reported findings provide the first examples that both 5'- and 3'-UTRs are essential and sufficient to drive differential translational regulation in a prokaryote and therefore have to functionally interact in vivo. The current results indicate that 3'-UTR-dependent translational control had already evolved before capping and polyadenylation of transcripts were invented, which are essential for circularization of transcripts in eukaryotes.

  13. Photoprotective function of chloroplast avoidance movement: in vivo chlorophyll fluorescence study.

    Science.gov (United States)

    Sztatelman, Olga; Waloszek, Andrzej; Banaś, Agnieszka Katarzyna; Gabryś, Halina

    2010-06-15

    Light-induced chloroplast avoidance movement has long been considered to be a photoprotective mechanism. Here, we present an experimental model in which this function can be shown for wild type Arabidopsis thaliana. We used blue light of different fluence rates for chloroplast positioning, and strong red light inactive in chloroplast positioning as a stressing light. The performance of photosystem II was measured by means of chlorophyll fluorescence. After stressing light treatment, a smaller decrease in photosystem II quantum yield was observed for leaves with chloroplasts in profile position as compared with leaves with chloroplasts in face position. Three Arabidopsis mutants, phot2 (no avoidance response), npq1 (impaired zeaxanhtin accumulation) and stn7 (no state transition), were examined for their chloroplast positioning and chlorophyll fluorescence parameters under identical experimental conditions. The results obtained for these mutants revealed additional stressing effects of blue light as compared with red light.

  14. A USPL functional system with articulated mirror arm for in-vivo applications in dentistry

    Science.gov (United States)

    Schelle, Florian; Meister, Jörg; Dehn, Claudia; Oehme, Bernd; Bourauel, Christoph; Frentzen, Mathias

    Ultra-short pulsed laser (USPL) systems for dental application have overcome many of their initial disadvantages. However, a problem that has not yet been addressed and solved is the beam delivery into the oral cavity. The functional system that is introduced in this study includes an articulated mirror arm, a scanning system as well as a handpiece, allowing for freehand preparations with ultra-short laser pulses. As laser source an Nd:YVO4 laser is employed, emitting pulses with a duration of tp cavity preparation are being demonstrated on mammoth ivory. This study indicates that freehand preparation employing an USPL system is possible but challenging, and accompanied by a variety of side-effects. The ablation rate with fixed handpiece is about 10 mm3/min. Factors like defocussing and blinding affect treatment efficiency. Laser sources with higher average output powers might be needed in order to reach sufficient preparation speeds.

  15. Dynamic noninvasive monitoring of renal function in vivo by fluorescence lifetime imaging

    Science.gov (United States)

    Goiffon, Reece J.; Akers, Walter J.; Berezin, Mikhail Y.; Lee, Hyeran; Achilefu, Samuel

    2009-03-01

    Kidneys normally filter the blood of excess salts and metabolic products, such as urea, while retaining plasma proteins. In diseases such as multiple myeloma and diabetes mellitus, the renal function is compromised and protein escapes into the urine. In this study, we present the use of fluorescence lifetime imaging (FLI) to image excess serum protein in urine (proteinuria). The near-infrared fluorescent dye LS-288 has distinct lifetimes when bound to protein versus free in solution, providing contrast between the protein-rich viscera and the mostly protein-free bladder. FLI with LS-288 in mice revealed that fluorescence lifetime (FLT) differences in the bladder relative to surrounding tissues was due to the fractional contributions of the bound and unbound dye molecules. The FLT of LS-288 decreased in the case of proteinuria while fluorescence intensity was unchanged. The results show that FLI can be useful for the dynamic imaging of protein-losing nephropathy due to diabetes mellitus and other renal diseases and suggest the potential use of the FLI to distinguish tumors from fluid-filled cysts in the body.

  16. Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo.

    Science.gov (United States)

    Laycock, Joseph E; Savoian, Matthew S; Glover, David M

    2006-06-01

    The metaphase-spindle steady-state length occurs as spindle microtubules ;flux', incorporating new subunits at their plus ends, while simultaneously losing subunits from their minus ends. Orbit/Mast/CLASP is required for tubulin subunit addition at kinetochores, and several kinesins regulate spindle morphology and/or flux by serving as microtubule depolymerases. Here, we use RNA interference in S2 cells to examine the relationship between Orbit and the four predicted kinesin-type depolymerases encoded by the Drosophila genome (Klp10A, Klp59C, Klp59D and Klp67A). Single depletion of Orbit results in monopolar spindles, mitotic arrest and a subsequent increase in apoptotic cells. These phenotypes are rescued by co-depleting Klp10A but none of the other three depolymerases. Spindle bipolarity is restored by preventing the spindle collapse seen in cells that lack Orbit, leading to functional spindles that are similar to controls in shape and length. We conclude that Klp10A exclusively antagonises Orbit in the regulation of bipolar spindle formation and maintenance.

  17. Rumen function in vivo and in vitro in sheep fed Leucaena leucocephala.

    Science.gov (United States)

    Barros-Rodríguez, Marcos Antonio; Solorio-Sánchez, Francisco Javier; Sandoval-Castro, Carlos Alfredo; Klieve, Athol; Rojas-Herrera, Rafael Antonio; Briceño-Poot, Eduardo Gaspar; Ku-Vera, Juan Carlos

    2015-04-01

    The effect of Leucaena leucocephala inclusion in sheep diets upon rumen function was evaluated. Nine Pelibuey sheep, 32.6 ± 5.33 kg live weight (LW), fitted with rumen cannula were used. A complete randomized block design was employed. Two experimental periods of 60 days each, with 60-day intervals between them, were used. Experimental treatments were as follows (n = 6): T1 (control), 100 % Pennisetum purpureum grass; T2, 20 % L. leucocephala + 80 % P. purpureum; T3, 40 % L. leucocephala + 60 % P. purpureum. In situ rumen neutral detergent fiber (aNDF) and crude protein (CP) degradation, dry matter intake (DMI), volatile fatty acids (VFA) production, estimated methane (CH4) yield, rumen pH, ammonia nitrogen (N-NH3), and protozoa counts were measured. The aNDF in situ rumen degradation of P. purpureum and leucaena was higher (P Leucaena CP degradation was higher in T2 and T3 but for P. purpureum it was only significantly higher in T3. Leucaena aNDF and CP degradation rate (c) was 50 % higher (P 0.05). Protozoa counts and in vitro gas production (48 h) were lower in T2 and T3 (P leucaena (P leucaena inclusion does increase rumen N-NH3, aNDF and CP digestibility, and voluntary intake.

  18. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.

    Science.gov (United States)

    Nelson, Christopher E; Hakim, Chady H; Ousterout, David G; Thakore, Pratiksha I; Moreb, Eirik A; Castellanos Rivera, Ruth M; Madhavan, Sarina; Pan, Xiufang; Ran, F Ann; Yan, Winston X; Asokan, Aravind; Zhang, Feng; Duan, Dongsheng; Gersbach, Charles A

    2016-01-22

    Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR-Cas9-based genome editing as a potential therapy to treat DMD. Copyright © 2016, American Association for the Advancement of Science.

  19. Tartrazine induces structural and functional aberrations and genotoxic effects in vivo.

    Science.gov (United States)

    Khayyat, Latifa; Essawy, Amina; Sorour, Jehan; Soffar, Ahmed

    2017-01-01

    Tartrazine is a synthetic organic azo dye widely used in food and pharmaceutical products. The current study aimed to evaluate the possible adverse effect of this coloring food additive on renal and hepatic structures and functions. Also, the genotoxic potential of tartrazine on white blood cells was investigated using comet assay. Twenty adult male Wistar rats were grouped into two groups of 10 each, control- and tartrazine-treated groups. The control group was administered orally with water alone. The experimental group was administered orally with tartrazine (7.5 mg/kg, b.wt.). Our results showed a marked increase in the levels of ALT, AST, ALP, urea, uric acid, creatinine, MDA and NO, and a decreased level of total antioxidants in the serum of rats dosed with tartrazine compared to controls. On the other hand, administration of tartrazine was associated with severe histopathological and cellular alterations of rat liver and kidney tissues and induced DNA damage in leucocytes as detected by comet assay. Taken together, the results showed that tartrazine intake may lead to adverse health effects.

  20. Tartrazine induces structural and functional aberrations and genotoxic effects in vivo

    Directory of Open Access Journals (Sweden)

    Latifa Khayyat

    2017-02-01

    Full Text Available Tartrazine is a synthetic organic azo dye widely used in food and pharmaceutical products. The current study aimed to evaluate the possible adverse effect of this coloring food additive on renal and hepatic structures and functions. Also, the genotoxic potential of tartrazine on white blood cells was investigated using comet assay. Twenty adult male Wistar rats were grouped into two groups of 10 each, control- and tartrazine-treated groups. The control group was administered orally with water alone. The experimental group was administered orally with tartrazine (7.5 mg/kg, b.wt.. Our results showed a marked increase in the levels of ALT, AST, ALP, urea, uric acid, creatinine, MDA and NO, and a decreased level of total antioxidants in the serum of rats dosed with tartrazine compared to controls. On the other hand, administration of tartrazine was associated with severe histopathological and cellular alterations of rat liver and kidney tissues and induced DNA damage in leucocytes as detected by comet assay. Taken together, the results showed that tartrazine intake may lead to adverse health effects.

  1. Identification of functionally important TonB-ExbD periplasmic domain interactions in vivo.

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    Ollis, Anne A; Postle, Kathleen

    2012-06-01

    In gram-negative bacteria, the cytoplasmic membrane proton-motive force energizes the active transport of TonB-dependent ligands through outer membrane TonB-gated transporters. In Escherichia coli, cytoplasmic membrane proteins ExbB and ExbD couple the proton-motive force to conformational changes in TonB, which are hypothesized to form the basis of energy transduction through direct contact with the transporters. While the role of ExbB is not well understood, contact between periplasmic domains of TonB and ExbD is required, with the conformational response of TonB to presence or absence of proton motive force being modulated through ExbD. A region (residues 92 to 121) within the ExbD periplasmic domain was previously identified as being important for TonB interaction. Here, the specific sites of periplasmic domain interactions between that region and the TonB carboxy terminus were identified by examining 270 combinations of 45 TonB and 6 ExbD individual cysteine substitutions for disulfide-linked heterodimer formation. ExbD residues A92C, K97C, and T109C interacted with multiple TonB substitutions in four regions of the TonB carboxy terminus. Two regions were on each side of the TonB residues known to interact with the TonB box of TonB-gated transporters, suggesting that ExbD positions TonB for correct interaction at that site. A third region contained a functionally important glycine residue, and the fourth region involved a highly conserved predicted amphipathic helix. Three ExbD substitutions, F103C, L115C, and T121C, were nonreactive with any TonB cysteine substitutions. ExbD D25, a candidate to be on a proton translocation pathway, was important to support efficient TonB-ExbD heterodimerization at these specific regions.

  2. 3D bioprinting of functional human skin: production and in vivo analysis.

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    Cubo, Nieves; Garcia, Marta; Del Cañizo, Juan F; Velasco, Diego; Jorcano, Jose L

    2016-12-05

    Significant progress has been made over the past 25 years in the development of in vitro-engineered substitutes that mimic human skin, either to be used as grafts for the replacement of lost skin, or for the establishment of in vitro human skin models. In this sense, laboratory-grown skin substitutes containing dermal and epidermal components offer a promising approach to skin engineering. In particular, a human plasma-based bilayered skin generated by our group, has been applied successfully to treat burns as well as traumatic and surgical wounds in a large number of patients in Spain. There are some aspects requiring improvements in the production process of this skin; for example, the relatively long time (three weeks) needed to produce the surface required to cover an extensive burn or a large wound, and the necessity to automatize and standardize a process currently performed manually. 3D bioprinting has emerged as a flexible tool in regenerative medicine and it provides a platform to address these challenges. In the present study, we have used this technique to print a human bilayered skin using bioinks containing human plasma as well as primary human fibroblasts and keratinocytes that were obtained from skin biopsies. We were able to generate 100 cm(2), a standard P100 tissue culture plate, of printed skin in less than 35 min (including the 30 min required for fibrin gelation). We have analysed the structure and function of the printed skin using histological and immunohistochemical methods, both in 3D in vitro cultures and after long-term transplantation to immunodeficient mice. In both cases, the generated skin was very similar to human skin and, furthermore, it was indistinguishable from bilayered dermo-epidermal equivalents, handmade in our laboratories. These results demonstrate that 3D bioprinting is a suitable technology to generate bioengineered skin for therapeutical and industrial applications in an automatized manner.

  3. Optically-Induced Neuronal Activity Is Sufficient to Promote Functional Motor Axon Regeneration In Vivo.

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    Patricia J Ward

    Full Text Available Peripheral nerve injuries are common, and functional recovery is very poor. Beyond surgical repair of the nerve, there are currently no treatment options for these patients. In experimental models of nerve injury, interventions (such as exercise and electrical stimulation that increase neuronal activity of the injured neurons effectively enhance axon regeneration. Here, we utilized optogenetics to determine whether increased activity alone is sufficient to promote motor axon regeneration. In thy-1-ChR2/YFP transgenic mice in which a subset of motoneurons express the light-sensitive cation channel, channelrhodopsin (ChR2, we activated axons in the sciatic nerve using blue light immediately prior to transection and surgical repair of the sciatic nerve. At four weeks post-injury, direct muscle EMG responses evoked with both optical and electrical stimuli as well as the ratio of these optical/electrical evoked EMG responses were significantly greater in mice that received optical treatment. Thus, significantly more ChR2+ axons successfully re-innervated the gastrocnemius muscle in mice that received optical treatment. Sections of the gastrocnemius muscles were reacted with antibodies to Synaptic Vesicle Protein 2 (SV2 to quantify the number of re-occupied motor endplates. The number of SV2+ endplates was greater in mice that received optical treatment. The number of retrogradely-labeled motoneurons following intramuscular injection of cholera toxin subunit B (conjugated to Alexa Fluor 555 was greater in mice that received optical treatment. Thus, the acute (1 hour, one-time optical treatment resulted in robust, long-lasting effects compared to untreated animals as well as untreated axons (ChR2-. We conclude that neuronal activation is sufficient to promote motor axon regeneration, and this regenerative effect is specific to the activated neurons.

  4. Formulation of Functionalized PLGA-PEG Nanoparticles for In Vivo Targeted Drug Delivery

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    Cheng, Jianjun; Teply, Benjamin A.; Sherifi, Ines; Sung, Josephine; Luther, Gaurav; Gu, Frank X.; Levy-Nissenbaum, Etgar; Radovic-Moreno, Aleksandar F.; Langer, Robert; Farokhzad, Omid C.

    2009-01-01

    Nanoparticle (NP) size has been shown to significantly effect the biodistribution of targeted and non-targeted NPs in an organ specific manner. Herein we have developed NPs from carboxy-terminated poly (d,l-lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG-COOH) polymer and studied the effects of altering the following formulation parameters on the size of NPs, including: 1) polymer concentration, 2) drug loading, 3) water miscibility of solvent, and 4) the ratio of water to solvent. We found that NP mean volumetric size correlates linearly with polymer concentration for NPs between 70 and 250 nm in diameter (linear coefficient = 0.99 for NPs formulated with solvents studied). NPs with desirable size, drug loading, and polydispersity were conjugated to the A10 RNA aptamer (Apt) that binds to the Prostate Specific Membrane Antigen (PSMA), and NP and NP-Apt biodistribution was evaluated in a LNCaP (PSMA+) xenograft mouse model of PCa. The surface functionalization of NPs with the A10 PSMA aptamer significantly enhanced delivery of NPs to tumors vs. equivalent NPs lacking the A10 PSMA aptamer (a 3.77-fold increase at 24 hrs; NP-Apt 0.83% ± 0.21% vs. NP 0.22% ± 0.07% of injected dose per gram of tissue; mean ± s.d., n = 4, p = 0.002). The ability to control NP size together with targeted delivery may result in favorable biodistribution and development of clinically relevant targeted therapies. PMID:17055572

  5. In vivo functional expression of a screened P. aeruginosa chaperone-dependent lipase in E. coli

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    Wu Xiangping

    2012-09-01

    Full Text Available Abstract Background Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA and its chaperone (LipB from a screened strain named AB which belongs to Pseudomonas aeruginosa were overexpressed in E. coli with two dual expression plasmid systems to enhance the production of the active lipase LipA without in vitro refolding process. Results In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as P. aeruginosa on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate the gene lipA (936 bp and lipase specific foldase gene lipB (1023 bp. One single expression plasmid system E. coli BL21/pET28a-lipAB and two dual expression plasmid systems E. coli BL21/pETDuet-lipA-lipB and E. coli BL21/pACYCDuet-lipA-lipB were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by E. coli BL21/pET28a-lipAB and E. coli BL21/pETDuet-lipA-lipB were 1300 U/L and 3200 U/L, respectively, while the activity of the lipase expressed by E. coli BL21/pACYCDuet-lipA-lipB was up to 8500 U/L. The lipase LipA had an optimal temperature of 30°C and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol

  6. Polysaccharides from Ganoderma formosanum function as a Th1 adjuvant and stimulate cytotoxic T cell response in vivo.

    Science.gov (United States)

    Pi, Chia-Chen; Chu, Ching-Liang; Lu, Chu-Ying; Zhuang, Yu-Jing; Wang, Cheng-Li; Yu, Yao-Hsuan; Wang, Hui-Yi; Lin, Chih-Chung; Chen, Chun-Jen

    2014-01-09

    The fungus of Ganoderma is a basidiomycete that possesses a variety of pharmacological effects and has been used in traditional Asian medicine for centuries. Ganoderma formosanum is a native Ganoderma species isolated in Taiwan, and we have previously demonstrated that PS-F2, a polysaccharide fraction purified from the submerged culture broth of G. formosanum, exhibits immunostimulatory properties in macrophages. In this study, we further characterized the adjuvant functions of PS-F2. In vitro, PS-F2 stimulated dendritic cells (DCs) to produce proinflammatory cytokines, including TNF-α, interleukin (IL)-6, and IL-12/IL-23 p40. PS-F2 also stimulated DCs to express the maturation markers CD40, CD80, CD86, and MHC class II. In a murine splenocyte culture, PS-F2 treatment resulted in elevated expression of T-bet and interferon (IFN)-γ in T lymphocytes. When used as an adjuvant in vivo with the ovalbumin (OVA) antigen, PS-F2 stimulated OVA-specific antibody production and primed IFN-γ production in OVA-specific T lymphocytes. PS-F2-adjuvated immunization also induced OVA-specific CTLs, which protected mice from a challenge with tumor cells expressing OVA. Collectively, our data show that PS-F2 functions as an adjuvant capable of inducing a Th1-polarized adaptive immune response, which would be useful in vaccines against viruses and tumors.

  7. Functional Assessment of Disease-Associated Regulatory Variants In Vivo Using a Versatile Dual Colour Transgenesis Strategy in Zebrafish

    Science.gov (United States)

    Bhatia, Shipra; Gordon, Christopher T.; Foster, Robert G.; Melin, Lucie; Abadie, Véronique; Baujat, Geneviève; Vazquez, Marie-Paule; Amiel, Jeanne; Lyonnet, Stanislas; van Heyningen, Veronica; Kleinjan, Dirk A.

    2015-01-01

    Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function. PMID:26030420

  8. Quantification of functional brace forces for posterior cruciate ligament injuries on the knee joint: an in vivo investigation.

    Science.gov (United States)

    LaPrade, Robert F; Smith, Sean D; Wilson, Katharine J; Wijdicks, Coen A

    2015-10-01

    Counteracting posterior translation of the tibia with an anterior force on the posterior proximal tibia has been demonstrated clinically to improve posterior knee laxity following posterior cruciate ligament (PCL) injury. This study quantified forces applied to the posterior proximal tibia by two knee braces designed for treatment of PCL injuries. The forces applied by two knee braces to the posterior proximal tibia and in vivo three-dimensional knee kinematics of six adult, male, healthy volunteer subjects (mean ± standard deviation: height, 182.5 ± 5.2 cm; body mass, 83.2 ± 9.3 kg; body mass index, 24.9 ± 1.5 kg/m(2); age, 25.8 ± 2.9 years) were measured using a custom pressure mapping technique and traditional surface marker motion capture techniques, while subjects performed three functional activities. The activities included seated unloaded knee flexion, squatting, and stair descent in a new generation dynamic force (DF) PCL brace and a static force (SF) PCL brace. During unloaded flexion at the lowest force level setting, the force applied by the DF brace increased as a function of flexion angle (slope = 0.7 N/°; p knee laxity following PCL injury. II.

  9. Pyrimidine motif triple helix in the Kluyveromyces lactis telomerase RNA pseudoknot is essential for function in vivo.

    Science.gov (United States)

    Cash, Darian D; Cohen-Zontag, Osnat; Kim, Nak-Kyoon; Shefer, Kinneret; Brown, Yogev; Ulyanov, Nikolai B; Tzfati, Yehuda; Feigon, Juli

    2013-07-02

    Telomerase is a ribonucleoprotein complex that extends the 3' ends of linear chromosomes. The specialized telomerase reverse transcriptase requires a multidomain RNA (telomerase RNA, TER), which includes an integral RNA template and functionally important template-adjacent pseudoknot. The structure of the human TER pseudoknot revealed that the loops interact with the stems to form a triple helix shown to be important for activity in vitro. A similar triple helix has been predicted to form in diverse fungi TER pseudoknots. The solution NMR structure of the Kluyveromyces lactis pseudoknot, presented here, reveals that it contains a long pyrimidine motif triple helix with unexpected features that include three individual bulge nucleotides and a C(+)•G-C triple adjacent to a stem 2-loop 2 junction. Despite significant differences in sequence and base triples, the 3D shape of the human and K. lactis TER pseudoknots are remarkably similar. Analysis of the effects of nucleotide substitutions on cell growth and telomere lengths provides evidence that this conserved structure forms in endogenously assembled telomerase and is essential for telomerase function in vivo.

  10. Cardiac magnetic resonance, transthoracic and transoesophageal echocardiography: a comparison of in vivo assessment of ventricular function in rats.

    Science.gov (United States)

    Richardson, J D; Bertaso, A G; Frost, L; Psaltis, P J; Carbone, A; Koschade, B; Wong, D T; Nelson, A J; Paton, S; Williams, K; Azarisman, S; Worthley, M I; Teo, K S; Gronthos, S; Zannettino, A C W; Worthley, S G

    2013-10-01

    In vivo assessment of ventricular function in rodents has largely been restricted to transthoracic echocardiography (TTE). However 1.5 T cardiac magnetic resonance (CMR) and transoesophageal echocardiography (TOE) have emerged as possible alternatives. Yet, to date, no study has systematically assessed these three imaging modalities in determining ejection fraction (EF) in rats. Twenty rats underwent imaging four weeks after surgically-induced myocardial infarction. CMR was performed on a 1.5 T scanner, TTE was conducted using a 9.2 MHz transducer and TOE was performed with a 10 MHz intracardiac echo catheter. Correlation between the three techniques for EF determination and analysis reproducibility was assessed. Moderate-strong correlation was observed between the three modalities; the greatest between CMR and TOE (intraclass correlation coefficient (ICC) = 0.89), followed by TOE and TTE (ICC = 0.70) and CMR and TTE (ICC = 0.63). Intra- and inter-observer variations were excellent with CMR (ICC = 0.99 and 0.98 respectively), very good with TTE (0.90 and 0.89) and TOE (0.87 and 0.84). Each modality is a viable option for evaluating ventricular function in rats, however the high image quality and excellent reproducibility of CMR offers distinct advantages even at 1.5 T with conventional coils and software.

  11. IL-7 modulates in vitro and in vivo human memory T regulatory cell functions through the CD39/ATP axis.

    Science.gov (United States)

    Younas, Mehwish; Hue, Sophie; Lacabaratz, Christine; Guguin, Aurélie; Wiedemann, Aurélie; Surenaud, Mathieu; Beq, Stéphanie; Croughs, Thérèse; Lelièvre, Jean-Daniel; Lévy, Yves

    2013-09-15

    The heterogeneity of human regulatory T cells (Tregs) may explain the discrepancies between studies on Tregs in physiology and pathology. Contrasting effects of IL-7 on the expansion and survival of human Tregs were reported. Therefore, we investigated the effects of IL-7 on the phenotype and function of well-characterized populations of human Tregs. We show that IL-7 signals via the CD127 receptor on naive, memory, and activated memory Tregs sorted from the blood of healthy donors, but it does not affect their proliferation. In contrast, IL-7 affects their suppressive capacities differently. This effect was modest on naive Tregs but was dramatic (90%) on memory Tregs. We provide evidence that IL-7 exerts a synergistic effect through downmodulation of the ectoenzyme CD39, which converts ATP to ADP/AMP, and an increase in ATP receptor P2X7. Both effects lead to an increase in the ATP-mediated effect, tipping the balance to favor Th17 conversion. Using an IL-7 therapeutic study, we show that IL-7 exerts the same effects in vitro and in vivo in HIV-infected individuals. Globally, our data show that IL-7 negatively regulates Tregs and contributes to increase the number of tools that may affect Treg function in pathology.

  12. In Vivo Transplantation of Enteric Neural Crest Cells into Mouse Gut; Engraftment, Functional Integration and Long-Term Safety.

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    Julie E Cooper

    Full Text Available Enteric neuropathies are severe gastrointestinal disorders with unsatisfactory outcomes. We aimed to investigate the potential of enteric neural stem cell therapy approaches for such disorders by transplanting mouse enteric neural crest cells (ENCCs into ganglionic and aganglionic mouse gut in vivo and analysing functional integration and long-term safety.Neurospheres generated from yellow fluorescent protein (YFP expressing ENCCs selected from postnatal Wnt1-cre;R26R-YFP/YFP murine gut were transplanted into ganglionic hindgut of wild-type littermates or aganglionic hindgut of Ednrbtm1Ywa mice (lacking functional endothelin receptor type-B. Intestines were then assessed for ENCC integration and differentiation using immunohistochemistry, cell function using calcium imaging, and long-term safety using PCR to detect off-target YFP expression.YFP+ ENCCs engrafted, proliferated and differentiated into enteric neurons and glia within recipient ganglionic gut. Transplanted cells and their projections spread along the endogenous myenteric plexus to form branching networks. Electrical point stimulation of endogenous nerve fibres resulted in calcium transients (F/F0 = 1.16 ± 0.01;43 cells, n = 6 in YFP+ transplanted ENCCs (abolished with TTX. Long-term follow-up (24 months showed transplanted ENCCs did not give rise to tumours or spread to other organs (PCR negative in extraintestinal sites. In aganglionic gut ENCCs similarly spread and differentiated to form neuronal and glial networks with projections closely associated with endogenous neural networks of the transition zone.Transplanted ENCCs successfully engrafted into recipient ganglionic and aganglionic gut showing appropriate spread, localisation and, importantly, functional integration without any long-term safety issues. This study provides key support for the development and use of enteric neural stem cell therapies.

  13. Alterations at the cross-bridge level are associated with a paradoxical gain of muscle function in vivo in a mouse model of nemaline myopathy.

    Science.gov (United States)

    Gineste, Charlotte; Ottenheijm, Coen; Le Fur, Yann; Banzet, Sébastien; Pecchi, Emilie; Vilmen, Christophe; Cozzone, Patrick J; Koulmann, Nathalie; Hardeman, Edna C; Bendahan, David; Gondin, Julien

    2014-01-01

    Nemaline myopathy is the most common disease entity among non-dystrophic skeletal muscle congenital diseases. The first disease causing mutation (Met9Arg) was identified in the gene encoding α-tropomyosin slow gene (TPM3). Considering the conflicting findings of the previous studies on the transgenic (Tg) mice carrying the TPM3Met9Arg mutation, we investigated carefully the effect of the Met9Arg mutation in 8-9 month-old Tg(TPM3)Met9Arg mice on muscle function using a multiscale methodological approach including skinned muscle fibers analysis and in vivo investigations by magnetic resonance imaging and 31-phosphorus magnetic resonance spectroscopy. While in vitro maximal force production was reduced in Tg(TPM3)Met9Arg mice as compared to controls, in vivo measurements revealed an improved mechanical performance in the transgenic mice as compared to the former. The reduced in vitro muscle force might be related to alterations occurring at the cross-bridges level with muscle-specific underlying mechanisms. In vivo muscle improvement was not associated with any changes in either muscle volume or energy metabolism. Our findings indicate that TPM3(Met9Arg) mutation leads to a mild muscle weakness in vitro related to an alteration at the cross-bridges level and a paradoxical gain of muscle function in vivo. These results clearly point out that in vitro alterations are muscle-dependent and do not necessarily translate into similar changes in vivo.

  14. Alterations at the cross-bridge level are associated with a paradoxical gain of muscle function in vivo in a mouse model of nemaline myopathy.

    Directory of Open Access Journals (Sweden)

    Charlotte Gineste

    Full Text Available Nemaline myopathy is the most common disease entity among non-dystrophic skeletal muscle congenital diseases. The first disease causing mutation (Met9Arg was identified in the gene encoding α-tropomyosin slow gene (TPM3. Considering the conflicting findings of the previous studies on the transgenic (Tg mice carrying the TPM3Met9Arg mutation, we investigated carefully the effect of the Met9Arg mutation in 8-9 month-old Tg(TPM3Met9Arg mice on muscle function using a multiscale methodological approach including skinned muscle fibers analysis and in vivo investigations by magnetic resonance imaging and 31-phosphorus magnetic resonance spectroscopy. While in vitro maximal force production was reduced in Tg(TPM3Met9Arg mice as compared to controls, in vivo measurements revealed an improved mechanical performance in the transgenic mice as compared to the former. The reduced in vitro muscle force might be related to alterations occurring at the cross-bridges level with muscle-specific underlying mechanisms. In vivo muscle improvement was not associated with any changes in either muscle volume or energy metabolism. Our findings indicate that TPM3(Met9Arg mutation leads to a mild muscle weakness in vitro related to an alteration at the cross-bridges level and a paradoxical gain of muscle function in vivo. These results clearly point out that in vitro alterations are muscle-dependent and do not necessarily translate into similar changes in vivo.

  15. In vivo functions of CPSF6 for HIV-1 as revealed by HIV-1 capsid evolution in HLA-B27-positive subjects.

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    Matthew S Henning

    2014-01-01

    Full Text Available The host protein CPSF6 possesses a domain that can interact with the HIV-1 capsid (CA protein. CPSF6 has been implicated in regulating HIV-1 nuclear entry. However, its functional significance for HIV-1 replication has yet to be firmly established. Here we provide evidence for two divergent functions of CPSF6 for HIV-1 replication in vivo. We demonstrate that endogenous CPSF6 exerts an inhibitory effect on naturally occurring HIV-1 variants in individuals carrying the HLA-B27 allele. Conversely, we find a strong selective pressure in these individuals to preserve CPSF6 binding, while escaping from the restrictive activity by CPSF6. This active maintenance of CPSF6 binding during HIV-1 CA evolution in vivo contrasts with the in vitro viral evolution, which can reduce CPSF6 binding to evade from CPSF6-mediated restriction. Thus, these observations argue for a beneficial role of CPSF6 for HIV-1 in vivo. CPSF6-mediated restriction renders HIV-1 less dependent or independent from TNPO3, RanBP2 and Nup153, host factors implicated in HIV-1 nuclear entry. However, viral evolution that maintains CPSF6 binding in HLA-B27+ subjects invariably restores the ability to utilize these host factors, which may be the major selective pressure for CPSF6 binding in vivo. Our study uncovers two opposing CA-dependent functions of CPSF6 in HIV-1 replication in vivo; however, the benefit for binding CPSF6 appears to outweigh the cost, providing support for a vital function of CPSF6 during HIV-1 replication in vivo.

  16. In vivo evidence of a functional association between immune cells in blood and brain in healthy human subjects.

    Science.gov (United States)

    Kanegawa, Naoki; Collste, Karin; Forsberg, Anton; Schain, Martin; Arakawa, Ryosuke; Jucaite, Aurelija; Lekander, Mats; Olgart Höglund, Caroline; Kosek, Eva; Lampa, Jon; Halldin, Christer; Farde, Lars; Varrone, Andrea; Cervenka, Simon

    2016-05-01

    Microglia, the resident macrophages in the central nervous system, are thought to be maintained by a local self-renewal mechanism. Although preclinical and in vitro studies have suggested that the brain may contain immune cells also from peripheral origin, the functional association between immune cells in the periphery and brain at physiological conditions is poorly understood. We examined 32 healthy individuals using positron emission tomography (PET) and [(11)C]PBR28, a radioligand for the 18-kDa translocator protein (TSPO) which is expressed both in brain microglia and blood immune cells. In 26 individuals, two measurements were performed with varying time intervals. In a subgroup of 19 individuals, of which 12 had repeat examinations, leukocyte numbers in blood was measured on each day of PET measurements. All individuals were genotyped for TSPO polymorphism and categorized as high, mixed, and low affinity binders. We assessed TSPO binding expressed as total distribution volume of [(11)C]PBR28 in brain and in blood cells. TSPO binding in brain was strongly and positively correlated to binding in blood cells both at baseline and when analyzing change between two PET examinations. Furthermore, there was a significant correlation between change of leukocyte numbers and change in TSPO binding in brain, and a trend-level correlation to change in TSPO binding in blood cells. These in vivo findings indicate an association between immunological cells in blood and brain via intact BBB, suggesting a functional interaction between these two compartments, such as interchange of peripherally derived cells or a common regulatory mechanism. Measurement of radioligand binding in blood cells may be a way to control for peripheral immune function in PET studies using TSPO as a marker of brain immune activation.

  17. Salmonid alphavirus replicon is functional in fish, mammalian and insect cells and in vivo in shrimps (Litopenaeus vannamei).

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    Olsen, Christel M; Pemula, Anand Kumar; Braaen, Stine; Sankaran, Krishnan; Rimstad, Espen

    2013-11-19

    The Salmonid alphavirus (SAV) is the etiological agent of pancreas disease in Atlantic salmon (Salmo salar) and Sleeping disease in rainbow trout (Oncorhynchus mykiss). SAV differs from alphaviruses infecting terrestrial animals in that it infects salmonid fish at low temperatures and does not use an arthropod vector for transmission. In this study we have shown that a SAVbased replicon could express proteins when driven by the subgenomic promoter in vitro in cells from fish, mammals and insects, as well as in vivo in shrimps (Litopanaeus vannamei). The SAV-replicon was found to be functional at temperatures ranging from 4 to 37°C. Protein expression was slow and moderate compared to that reported from terrestrial alphavirus replicons or from vectors where protein expression was under control of the immediate early CMV-promoter. No cytopathic effect was visually observable in cells transfected with SAV-replicon vectors. Double stranded RNA was present for several days after transfection of the SAV-replicon in fish cell lines and its presence was indicated also in shrimp. The combination of prolonged dsRNA production, low toxicity, and wide temperature range for expression, may potentially be advantageous for the use of the SAV replicon to induce immune responses in aquaculture of fish and shrimp.

  18. Probe-hosted silicon photomultipliers for time-domain functional near-infrared spectroscopy: phantom and in vivo tests.

    Science.gov (United States)

    Re, Rebecca; Martinenghi, Edoardo; Mora, Alberto Dalla; Contini, Davide; Pifferi, Antonio; Torricelli, Alessandro

    2016-10-01

    We report the development of a compact probe for time-domain (TD) functional near-infrared spectroscopy (fNIRS) based on a fast silicon photomultiplier (SiPM) that can be put directly in contact with the sample without the need of optical fibers for light collection. We directly integrated an avalanche signal amplification stage close to the SiPM, thus reducing the size of the detection channel and optimizing the signal immunity to electromagnetic interferences. The whole detection electronics was placed in a plastic screw holder compatible with the electroencephalography standard cap for measurement on brain or with custom probe holders. The SiPM is inserted into a transparent and insulating resin to avoid the direct contact of the scalp with the 100-V bias voltage. The probe was integrated in an instrument for TD fNIRS spectroscopy. The system was characterized on tissue phantoms in terms of temporal resolution, responsivity, linearity, and capability to detect deep absorption changes. Preliminary in vivo tests on adult volunteers were performed to monitor hemodynamic changes in the arm during a cuff occlusion and in the brain cortex during a motor task.

  19. BMP2-encapsulated chitosan coatings on functionalized Ti surfaces and their performance in vitro and in vivo.

    Science.gov (United States)

    Han, Lu; Lin, Hong; Lu, Xiong; Zhi, Wei; Wang, Ke-Feng; Meng, Fan-Zhi; Jiang, Ou

    2014-07-01

    Bone morphogenic protein-2 (BMP2)-encapsulated chitosan (CS) coatings were prepared to immobilize BMP2 on titanium (Ti) surfaces. The Ti substrates were functionalized through a three-step process: alkali treatment, silanization with 3-aminopropyltriethoxysilane and aldehydation with glutaraldehyde (GA). BMP2-encapsulated CS coatings (BMP2-CS) were bonded to Ti surfaces through reactions between the aldehyde groups of GA and the amine groups of CS. Direct BMP2 immobilization on aldehyde-treated Ti (BMP2-Ti) and pure CS coatings (CS-Ti) were used as controls. The release rate of BMP2-CS-Ti was half of that of BMP2-Ti at initial stage, which indicates that the CS coatings are suitable carriers for sustained BMP2 release. The osteoinductivities of BMP2-CS-Ti, BMP2-Ti, CS-Ti and pristine Ti were examined by both in vitro cell tests and in vivo experiments. Bone marrow stem cell (BMSC) culture indicated that BMP2-CS-Ti is more potent in stimulating the differentiation of the adhering BMSC than the three other groups. Rabbit femur implantation revealed the excellent osteoinductivity of BMP2-CS-coated Ti implants. These results demonstrate that the BMP2-encapsulated CS coatings are stable osteoinductive coatings that realize the sustained release of BMP2 and maintain the activity of the protein.

  20. Murine T cell clones specific for Hymenolepis nana: generation and functional analysis in vivo and in vitro.

    Science.gov (United States)

    Asano, K; Okamoto, K

    1991-12-01

    To examine the role of the T cell in protective immunity to Hymenolepis nana, H. nana-specific clonal lymphocytes were generated from mesenteric lymph nodes of BALB/c mice infected with H. nana, and some of their functions were analyzed in vitro and in vivo. Following limiting dilution techniques, five clones were generated from mesenteric lymph node cell populations. All of these clones expressed the L3T4+, Lyt-2.2- phenotype and proliferated in vitro in response to soluble egg antigen of H. nana. Of five clones, three secreted interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) after stimulation with egg antigen. Furthermore, these three clones conferred local delayed-type hypersensitivity to egg antigen. The remaining two clones produced interleukin 4 (IL-4) in response to egg antigen, and could not mediate local delayed-type hypersensitivity. Adoptive transfer experiments using clonal lymphocytes were also undertaken in an attempt to define cell types involved in protective immunity. Clonal lymphocytes secreting both IL-2 and IFN-gamma transferred protective immunity, equivalent to that obtained by non-cultured-sensitized mesenteric lymph node cells. They were effective in very small numbers. However, clonal lymphocytes that secreted IL-4 did not transfer protective immunity. These results suggest that helper T lymphocytes, especially the Th1 subtype, are involved in protective immunity against H. nana.

  1. Cholecystokinin rapidly stimulates CrkII function in vivo in rat pancreatic acini. Formation of CrkII-protein complexes.

    Science.gov (United States)

    Andreolotti, Alberto G; Bragado, Maria J; Tapia, Jose A; Jensen, Robert T; Garcia-Marin, Luis J

    2003-12-01

    Crk belongs to a family of adapter proteins whose structure allows interaction with tyrosine-phosphorylated proteins and is therefore an important modulator of downstream signals, representing a convergence of the actions of numerous stimuli. Recently, it was demonstrated that cholecystokinin (CCK) induced tyrosine phosphorylation of proteins related to fiber stress formation in rat pancreatic acini. Here, we investigated whether CCK receptor activation signals through CrkII and forms complexes with tyrosine-phosphorylated proteins in rat pancreatic acini. We demonstrated that CCK promoted the transient formation of CrkII-paxillin and CrkII-p130Cas complexes with maximal effect at 1 min. Additionally, CCK decreased the electrophoretic mobility of CrkII. This decrease was time- and concentration-dependent and inversely related with its function. Carbachol and bombesin also decreased CrkII electrophoretic mobility, whereas epidermal growth factor, vasoactive intestinal peptide, secretin or pituitary adenylate cyclase-activating polypeptide had no effect. CCK-induced CrkII electrophoretic shift was dependent on the Src family of tyrosine kinases and occurred in the intact animal, suggesting a physiological role of CrkII mediating CCK actions in the exocrine pancreas in vivo.

  2. SURFACE MULTI-FUNCTIONALIZATION OF POLY(LACTIC ACID)NANOPARTICLES AND C6 GLIOMA CELL TARGETING in vivo

    Institute of Scientific and Technical Information of China (English)

    Xu-bo Yuan; Chun-sheng Kang; Yun-hui Zhao; Ming-qi Gu; Pei-yu Pu

    2009-01-01

    Polysaccharide coated PLA nanopartieles bearing aldehyde groups were prepared by dialysis of DMSO solution of cholesterol hydrophobic-modified dextran polyaldehyde and PLA against water.The average diameter of the nanoparticles was about 160 nm,and the size distribution was nearly homogenous.The nanoparticles were functionalized simultaneously with CD71 and EGFR antibody through the Schiff's base reaction,and then radiolabeled with 99mTc.After perfused the radiolabeled nanoparticles into tumor-bearing rats through left common carotid artery,the radioactivity in liver,spleen,kidney and brain was measured by scintillation counter.The results showed that less than 2% of nanoparticles were uptaken by the brain due to the uptake of the nanoparticles by the RES system.However,the coupling of transferrin antibody on the nanoparticles facilitated the penetration of nanoparticles across the blood brain barrier,and more specially,compared with monofuctionalized and native nanoparticles,the multifunctionalization enhanced the tumor accumulation of the nanoparticles in vivo.

  3. Dual-Functional Polyethylene Glycol-b-polyhexanide Surface Coating with in Vitro and in Vivo Antimicrobial and Antifouling Activities.

    Science.gov (United States)

    Zhi, Zelun; Su, Yajuan; Xi, Yuewei; Tian, Liang; Xu, Miao; Wang, Qianqian; Padidan, Sara; Li, Peng; Huang, Wei

    2017-03-29

    In recent years, microbial colonization on the surface of biomedical implants/devices has become a severe threat to human health. Herein, surface-immobilized guanidine derivative block copolymers create an antimicrobial and antifouling dual-functional coating. We report the preparation of an antimicrobial and antifouling block copolymer by the conjugation of polyhexanide (PHMB) with either allyl glycidyl ether or allyloxy polyethylene glycol (APEG; MW 1200 and 2400). The allyl glycidyl ether modified PHMB (A-PHMB) and allyloxy polyethylene glycol1200/2400 modified PHMB (APEG1200/2400-PHMB) copolymers were grafted onto a silicone rubber surface as a bottlebrush-like coating, respectively, using a plasma-UV-assisted surface-initiated polymerization. Both A-PHMB and APEG1200/2400-PHMB coatings exhibited excellent broad-spectrum antimicrobial properties against Gram-negative/positive bacteria and fungi. The APEG2400-PHMB coating displayed an improved antibiofilm as well as antifouling properties and a long reusable cycle, compared with two other coatings, due to its abundant PEG blocks among those copolymers. Also, the APEG2400-PHMB-coated silicone coupons were biocompatible toward mammalian cells, as revealed by in vitro hemocompatibile and cytotoxic assays. An in vivo study showed a significant decline of Escherichia coli colonies with a 5-log reduction, indicating the APEG2400-PHMB coating surface worked effectively in the rodent subcutaneous infection model. This PHMB-based block copolymer coating is believed to be an effective strategy to prevent biomaterial-associated infections.

  4. A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth in vivo

    Directory of Open Access Journals (Sweden)

    Powers David

    2007-11-01

    Full Text Available Abstract Background Integrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration. The integrin α5β1 has been shown to play a critical role during angiogenesis. An inhibitor of this integrin, volociximab (M200, inhibits endothelial cell growth and movement in vitro, independent of the growth factor milieu, and inhibits tumor growth in vivo in the rabbit VX2 carcinoma model. Although volociximab has already been tested in open label, pilot phase II clinical trials in melanoma, pancreatic and renal cell cancer, evaluation of the mechanism of action of volociximab has been limited because this antibody does not cross-react with murine α5β1, precluding its use in standard mouse xenograft models. Methods We generated a panel of rat-anti-mouse α5β1 antibodies, with the intent of identifying an antibody that recapitulated the properties of volociximab. Hybridoma clones were screened for analogous function to volociximab, including specificity for α5β1 heterodimer and blocking of integrin binding to fibronectin. A subset of antibodies that met these criteria were further characterized for their capacities to bind to mouse endothelial cells, inhibit cell migration and block angiogenesis in vitro. One antibody that encompassed all of these attributes, 339.1, was selected from this panel and tested in xenograft models. Results A panel of antibodies was characterized for specificity and potency. The affinity of antibody 339.1 for mouse integrin α5β1 was determined to be 0.59 nM, as measured by BIAcore. This antibody does not significantly cross-react with human integrin, however 339.1 inhibits murine endothelial cell migration and tube formation and elicits cell death in these cells (EC50 = 5.3 nM. In multiple xenograft models, 339.1 inhibited the growth of established tumors by 40–60% (p Conclusion The results herein demonstrate that 339.1, like volociximab, exhibits potent anti-α5β1

  5. Genetic reduction of mitochondrial complex I function does not lead to loss of dopamine neurons in vivo.

    Science.gov (United States)

    Kim, Hyung-Wook; Choi, Won-Seok; Sorscher, Noah; Park, Hyung Joon; Tronche, François; Palmiter, Richard D; Xia, Zhengui

    2015-09-01

    Inhibition of mitochondrial complex I activity is hypothesized to be one of the major mechanisms responsible for dopaminergic neuron death in Parkinson's disease. However, loss of complex I activity by systemic deletion of the Ndufs4 gene, one of the subunits comprising complex I, does not cause dopaminergic neuron death in culture. Here, we generated mice with conditional Ndufs4 knockout in dopaminergic neurons (Ndufs4 conditional knockout mice [cKO]) to examine the effect of complex I inhibition on dopaminergic neuron function and survival during aging and on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in vivo. Ndufs4 cKO mice did not show enhanced dopaminergic neuron loss in the substantia nigra pars compacta or dopamine-dependent motor deficits over the 24-month life span. These mice were just as susceptible to MPTP as control mice. However, compared with control mice, Ndufs4 cKO mice exhibited an age-dependent reduction of dopamine in the striatum and increased α-synuclein phosphorylation in dopaminergic neurons of the substantia nigra pars compacta. We also used an inducible Ndufs4 knockout mouse strain (Ndufs4 inducible knockout) in which Ndufs4 is conditionally deleted in all cells in adult to examine the effect of adult onset, complex I inhibition on MPTP sensitivity of dopaminergic neurons. The Ndufs4 inducible knockout mice exhibited similar sensitivity to MPTP as control littermates. These data suggest that mitochondrial complex I inhibition in dopaminergic neurons does contribute to dopamine loss and the development of α-synuclein pathology. However, it is not sufficient to cause cell-autonomous dopaminergic neuron death during the normal life span of mice. Furthermore, mitochondrial complex I inhibition does not underlie MPTP toxicity in vivo in either cell autonomous or nonautonomous manner. These results provide strong evidence that inhibition of mitochondrial complex I activity is not sufficient to cause dopaminergic neuron

  6. Cocaine- and amphetamine-regulated transcript (CART) peptide as an in vivo regulator of cardiac function in Rana ridibunda frog

    National Research Council Canada - National Science Library

    Iliyana V. Ivanova; Rudolf Schubert; Dessislava B. Duridanova; Thomas B. Bolton; Lubomir T. Lubomirov; Hristo S. Gagov

    2007-01-01

    The aim of this study was to investigate the effect of CART peptide on cardiac performance and on the physiological signalling pathways involved using Rana ridibunda frog heart preparations in vivo...

  7. A novel single walled carbon nanotube (SWCNT) functionalization agent facilitating in vivo combined chemo/thermo therapy

    Science.gov (United States)

    Zhang, Liwen; Rong, Pengfei; Chen, Minglong; Gao, Shi; Zhu, Lei

    2015-10-01

    Carbon nanotubes (CNTs) have shown intriguing applications in biotechnological and biomedical fields due to their unique shape and properties. However, the fact that unmodified CNTs are prone to aggregation, stunts CNTs applications under physiological conditions. In this research, we found that as little as 1/5th the single walled carbon nanotube (SWCNT) weight of Evans Blue (EB) is capable of dispersing SWCNT as well as facilitating SWCNT functionalization. In view of the binding between EB and albumin, the yielding product (SWCNT/EB) demonstrated extreme stability for weeks under physiological conditions and it can be endowed with a therapeutic ability by simply mixing SWCNT/EB with an albumin based drug. Specifically, the formed SWCNT/EB/albumin/PTX nanocomplex exhibits strong near-infrared (NIR) absorbance, and can serve as an agent for chemo/thermal therapeutic purposes. Our in vivo result reveals that SWCNT/EB/albumin/PTX after being administered into the MDA-MB-435 tumor would effectively ablate the tumor by chemo and photothermal therapy. Such a combined treatment strategy provides remarkable therapeutic outcomes in restraining tumor growth compared to chemo or photothermal therapy alone. Overall, our strategy of dispersing SWCNTs by EB can be used as a platform for carrying other drugs or functional genes with the aid of albumin to treat diseases. The present study opens new opportunities in surface modification of SWCNTs for future clinical disease treatment.Carbon nanotubes (CNTs) have shown intriguing applications in biotechnological and biomedical fields due to their unique shape and properties. However, the fact that unmodified CNTs are prone to aggregation, stunts CNTs applications under physiological conditions. In this research, we found that as little as 1/5th the single walled carbon nanotube (SWCNT) weight of Evans Blue (EB) is capable of dispersing SWCNT as well as facilitating SWCNT functionalization. In view of the binding between EB and

  8. Studies on the function of hepatic lipase in the cat after immunological blockade of the enzyme in vivo.

    Science.gov (United States)

    Demacker, P N; Hijmans, A G; Stalenhoef, A F; van 't Laar, A

    1988-02-01

    In order to investigate the in vivo function of hepatic lipase, cats were injected with anti-cat hepatic lipase antibodies which produced a complete and specific inhibition of heparin-releasable hepatic lipase. The cat was chosen as an animal model because it displays, like man, a relative deficiency of lipoprotein lipase compared to hepatic lipase and because the possession of two subfractions of high density lipoproteins, HDL2 and HDL3. In fasted cats no changes were observed in plasma triglycerides or phospholipids. In fed animals triglycerides increased considerably, indicating that hepatic lipase may have a function in the postprandial phase. In fat-loaded cats (6 g of fat/kg) triglycerides in the d less than 1.019 g/ml fraction increased from 4 h after the blockade due to accumulation of lipoproteins with pre-beta-mobility containing the apoproteins, apo B-100, apo E and apo A-I. Apo B-48 did not accumulate consistently. Phospholipids in the HDL2-fraction and those in the HDL3-fraction of the fat-loaded cats tended to increase and decrease from 6 and 9 h after the blockade, respectively. The absolute change in HDL2 phospholipids approximated that of HDL3-phospholipids. Overall, the density of HDL particles decreased, apparently secondary to the accumulation of apo A-I in the d less than 1.019 g/ml fraction. Our findings suggest that hepatic lipase is involved in the hydrolysis of a special class of apo A-I containing triglyceride-rich lipoproteins synthesised in the postprandial phase.

  9. Persistent Borna Disease Virus infection changes expression and function of astroglial gap junctions in vivo and in vitro.

    Science.gov (United States)

    Köster-Patzlaff, Christiane; Hosseini, Seyed Mehdi; Reuss, Bernhard

    2007-12-12

    Neonatal Borna Disease Virus (BDV) infection of the Lewis rat brain leads to dentate gyrus (DG) degeneration, underlying mechanisms are not fully understood. Since astroglial gap junction (GJ) coupling is known to influence neurodegenerative processes, the question arose whether persistent BDV infection influences astroglial connexins (Cx) Cx43 and Cx30 in the hippocampal formation (HiF) of Lewis rats. RT-PCR and Western blot analysis of forebrain (FB) samples revealed a virus dependent reduction of both Cx types 8 but not 4 weeks post infection (p.i.). Immunohistochemistry revealed an increase of Cx43 in the DG and a decrease in the CA3 region 4 and 8 weeks p.i. Cx30, which was detectable only 8 weeks p.i., revealed a BDV dependent increase in DG and CA3 regions. BDV dependent astrogliosis as revealed by immunodetection of glial fibrillary acidic protein (GFAP) correlated not with astroglial connexin expression. With regard to functional coupling as revealed by scrape loading, BDV infection resulted in increased spreading of the GJ permeant dye Lucifer yellow in primary hippocampal astroglial cultures, and in increased expression of Cx43 and Cx30 as revealed by immunocytochemistry. In conclusion, persistent BDV infection of the Lewis rat brain leads to changes in astroglial Cx expression both in vivo and in vitro and of functional coupling in vitro. Distribution and time course of these changes suggest them to be a direct result of neurodegeneration in the DG and an indirect effect of neuronal deafferentiation in the CA3 region.

  10. Functional mapping imprinted quantitative trait loci underlying developmental characteristics

    Directory of Open Access Journals (Sweden)

    Li Gengxin

    2008-03-01

    Full Text Available Abstract Background Genomic imprinting, a phenomenon referring to nonequivalent expression of alleles depending on their parental origins, has been widely observed in nature. It has been shown recently that the epigenetic modification of an imprinted gene can be detected through a genetic mapping approach. Such an approach is developed based on traditional quantitative trait loci (QTL mapping focusing on single trait analysis. Recent studies have shown that most imprinted genes in mammals play an important role in controlling embryonic growth and post-natal development. For a developmental character such as growth, current approach is less efficient in dissecting the dynamic genetic effect of imprinted genes during individual ontology. Results Functional mapping has been emerging as a powerful framework for mapping quantitative trait loci underlying complex traits showing developmental characteristics. To understand the genetic architecture of dynamic imprinted traits, we propose a mapping strategy by integrating the functional mapping approach with genomic imprinting. We demonstrate the approach through mapping imprinted QTL controlling growth trajectories in an inbred F2 population. The statistical behavior of the approach is shown through simulation studies, in which the parameters can be estimated with reasonable precision under different simulation scenarios. The utility of the approach is illustrated through real data analysis in an F2 family derived from LG/J and SM/J mouse stains. Three maternally imprinted QTLs are identified as regulating the growth trajectory of mouse body weight. Conclusion The functional iQTL mapping approach developed here provides a quantitative and testable framework for assessing the interplay between imprinted genes and a developmental process, and will have important implications for elucidating the genetic architecture of imprinted traits.

  11. In vivo venous assessment of red blood cell aggregate sizes in diabetic patients with a quantitative cellular ultrasound imaging method: proof of concept.

    Directory of Open Access Journals (Sweden)

    Julien Tripette

    Full Text Available Diabetic patients present higher level of red blood cell (RBC aggregation contributing to the development of vascular complications. While it has been suggested that this hematology/rheology parameter could bring additional prognostic information for the management of those patients, RBC aggregation screening is not included as a clinical practice. Most medical centers are not equipped to measure properly this parameter, although sedimentation tests can bring some indication. Here, we aimed at evaluating the feasibility of using ultrasound to assess in-vivo hyper-aggregation in type 2 diabetic patients.Seventeen diabetic patients and 15 control subjects underwent ultrasound measurements of RBC aggregation in both cephalic and great saphenous veins. Non-invasive in-vivo ultrasound measurements were performed using a newly developed cellular imaging technique, the structure factor size and attenuation estimator (SFSAE. Comparisons with an ex-vivo gold standard rheometry technique were done, along with measurements of pro-aggregating plasma molecule concentrations.In-vivo RBC aggregation was significantly higher in diabetic patients compared with controls for cephalic vein measurements, while a trend (p = 0.055 was noticed in the great saphenous vein. SFSAE measurements were correlated with gold standard in-vitro measures, fibrinogen and C-reactive protein plasma concentrations.RBC aggregation can be measured in-vivo in diabetic patients using ultrasound. Prospective studies are needed to determine whether the SFSAE method could help clinicians in the early management of vascular complications in this patient population.

  12. Functional analysis of zebrafish microfibril-associated glycoprotein-1 (Magp1) in vivo reveals roles for microfibrils in vascular development and function.

    Science.gov (United States)

    Chen, Eleanor; Larson, Jon D; Ekker, Stephen C

    2006-06-01

    Mutations in fibrillin-1 (FBN1) result in Marfan syndrome, demonstrating a critical requirement for microfibrils in vessel structure and function. However, the identity and function of many microfibril-associated molecules essential for vascular development and function have yet to be characterized. In our morpholino-based screen for members of the secretome required for vascular development, we identified a key player in microfibril formation in zebrafish embryogenesis. Microfibril-associated glycoprotein-1 (MAGP1) is a conserved protein found in mammalian and zebrafish microfibrils. Expression of magp1 mRNA is detected in microfibril-producing cells. Analysis of a functional Magp1-mRFP fusion protein reveals localization along the midline and in the vasculature during embryogenesis. Underexpression and overexpression analyses demonstrate that specific Magp1 protein levels are critical for vascular development. Integrin function is compromised in magp1 morphant embryos, suggesting that reduced integrin-matrix interaction is the main mechanism for the vascular defects in magp1 morphants. We further show that Magp1 and fibrillin-1 interact in vivo. This study implicates MAGP1 as a key player in microfibril formation and integrity during development. The essential role for MAGP1 in vascular morphogenesis and function also supports a wide range of clinical applications, including therapeutic targets in vascular disease and cardiovascular tissue engineering.

  13. Pharmaceutical evaluation of naftopidil enantiomers: Rat functional assays in vitro and estrogen/androgen induced rat benign prostatic hyperplasia model in vivo.

    Science.gov (United States)

    Huang, Jun-Jun; Cai, Yi; Huang, Min-Yi; Zhu, Liu; He, Fei; Liu, Xia-Wen; Huang, Bi-Yun; Yi, Yan-Zhen; Yuan, Mu

    2016-11-15

    Naftopidil (NAF) is a α1D/1A adrenoceptor selective drug used for the treatment of both benign prostatic hyperplasia and lower urinary tract symptoms (BPH/LUTS). However, NAF is used as a racemate in clinic. To compare the differences and similarities among two enantiomers and racemate, pharmacological activities were evaluated through rat functional assays in vitro and estrogen/androgen (E/T) induced rat BPH model in vivo. NAF and the two enantiomers showed similar blocking activity on α1 receptor. S-NAF exhibited more α1D/1A adrenoceptor subtype selectivity than R-NAF and the racemate. The selectivity ratios pA2 (α1D)/pA2 (α1B) and pA2 (α1A)/pA2 (α1B) were 40.7- and 16.2-fold, respectively. NAF and its enantiomers effectively prevented the development of rat prostatic hyperplasia via suppressing the increase of the prostatic wet weight, visually. The quantitative analysis of the relative acinus volume, relative stroma volume, relative epithelial volume, epithelial height and expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were carried out. S-NAF showed an advantage on the effect of inhibiting prostate wet weight and stroma volume over R-NAF and racemate NAF (P<0.05). Nevertheless, no other significant difference was observed between these two enantiomers. In conclusion, both R-NAF and S-NAF not only relax prostate muscle but also inhibit the prostate growth, thus relieve BPH.

  14. Autoimmune vitiligo is associated with gain-of-function by a transcriptional regulator that elevates expression of HLA-A*02:01 in vivo.

    Science.gov (United States)

    Hayashi, Masahiro; Jin, Ying; Yorgov, Daniel; Santorico, Stephanie A; Hagman, James; Ferrara, Tracey M; Jones, Kenneth L; Cavalli, Giulio; Dinarello, Charles A; Spritz, Richard A

    2016-02-02

    HLA-A is a class I major histocompatibility complex receptor that presents peptide antigens on the surface of most cells. Vitiligo, an autoimmune disease in which skin melanocytes are destroyed by cognate T cells, is associated with variation in the HLA-A gene; specifically HLA-A*02:01, which presents multiple vitiligo melanocyte autoantigens. Refined genetic mapping localizes vitiligo risk in the HLA-A region to an SNP haplotype ∼20-kb downstream, spanning an ENCODE element with many characteristics of a transcriptional enhancer. Convergent CTCF insulator sites flanking the HLA-A gene promoter and the predicted transcriptional regulator, with apparent interaction between these sites, suggests this element regulates the HLA-A promoter. Peripheral blood mononuclear cells from healthy subjects homozygous for the high-risk haplotype expressed 39% more HLA-A RNA than cells from subjects carrying nonhigh-risk haplotypes (P = 0.0048). Similarly, RNAseq analysis of 1,000 Genomes Project data showed more HLA-A mRNA expressed in subjects homozygous for the high-risk allele of lead SNP rs60131261 than subjects homozygous for the low-risk allele (P = 0.006). Reporter plasmid transfection and genomic run-on sequence analyses confirm that the HLA-A transcriptional regulator contains multiple bidirectional promoters, with greatest activity on the high-risk haplotype, although it does not behave as a classic enhancer. Vitiligo risk associated with the MHC class I region thus derives from combined quantitative and qualitative phenomena: a SNP haplotype in a transcriptional regulator that induces gain-of-function, elevating expression of HLA-A RNA in vivo, in strong linkage disequilibrium with an HLA-A allele that confers *02:01 specificity.

  15. Complete in vivo reversal of P-glycoprotein pump function in the blood-brain barrier visualized with positron emission tomography

    NARCIS (Netherlands)

    Hendrikse, NH; Schinkel, AH; De Vries, EGE; Fluks, E; Van der Graaf, WTA; Willemsen, ATM; Vaalburg, W; Franssen, EJF

    1998-01-01

    1 Homozygously mdr1a gene disrupted mice (mdr1a(-/-) mice) and wild type mice (mdr1a(+/+) mice) were used to develop a method for P-glycoprotein (P-gp) function imaging non-invasively and to study the effect of a P-gp reversal agent on its function in vivo. 2 [C-11]verapamil (0.1 mg/kg) was administ

  16. Heterogeneous abnormalities of in-vivo left ventricular calcium influx and function in mouse models of muscular dystrophy cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Greally Elizabeth

    2013-01-01

    Full Text Available Abstract Background Manganese-enhanced cardiovascular magnetic resonance (MECMR can non-invasively assess myocardial calcium influx, and calcium levels are known to be elevated in muscular dystrophy cardiomyopathy based on cellular studies. Methods Left ventricular functional studies and MECMR were performed in mdx mice (model of Duchenne Muscular Dystrophy, 24 and 40 weeks and Sgcd−/− mice (Limb Girdle Muscular Dystrophy 2 F, 16 and 32 weeks, compared to wild type controls (C57Bl/10, WT. Results Both models had left ventricular hypertrophy at the later age compared to WT, though the mdx mice had reduced stroke volumes and the Sgcd−/− mice increased heart rate and cardiac index. Especially at the younger ages, MECMR was significantly elevated in both models (both Pmdx mice (PSgcd−/− mice (PSgcd−/− mice had increased heart rates, to determine the role of heart rate in MECMR we studied the hyperpolarization-activated cyclic nucleotide-gated channel inhibitor ZD 7288 which selectively reduces heart rate. This reduced heart rate and MECMR in all mouse groups. However, when looking at the time course of reduction of MECMR in the Sgcd−/− mice at up to 5 minutes of the manganese infusion when heart rates were matched to the WT mice, MECMR was still significantly elevated in the Sgcd−/− mice (P Conclusions Despite both mouse models exhibiting increased in-vivo calcium influx at an early stage in the development of the cardiomyopathy before left ventricular hypertrophy, there are distinct phenotypical differences between the 2 models in terms of heart rates, hemodynamics and responses to calcium channel inhibitors.

  17. In vivo biocompatibility and pacing function study of silver ion-based antimicrobial surface technology applied to cardiac pacemakers

    Science.gov (United States)

    Shawcross, James; Bakhai, Ameet; Ansaripour, Ali; Armstrong, James; Lewis, David; Agg, Philip; De Godoy, Roberta; Blunn, Gordon

    2017-01-01

    Introduction Evidence suggests that the rate of cardiovascular implantable electronic device (CIED) infections is increasing more rapidly than the rates of CIED implantation and is associated with considerable mortality, morbidity and health economic impact. Antimicrobial surface treatments are being developed for CIEDs to reduce the risk of postimplantation infection within the subcutaneous implant pocket. Methods and analysis The feasibility of processing cardiac pacemakers with the Agluna antimicrobial silver ion surface technology and in vivo biocompatibility were evaluated. Antimicrobially processed (n=6) and control pacemakers (n=6) were implanted into subcutaneous pockets and connected to a part of the sacrospinalis muscle using an ovine model for 12 weeks. Pacemaker function was monitored preimplantation and postimplantation. Results Neither local infection nor systemic toxicity were detected in antimicrobial or control devices, and surrounding tissues showed no abnormal pathology or over-reactivity. Semiquantitative scores of membrane formation, cellular orientation and vascularity were applied over five regions of the pacemaker capsule and average scores compared. Results showed no significant difference between antimicrobially processed and control pacemakers. Silver analysis of whole blood at 7 days found that levels were a maximum of 10 parts per billion (ppb) for one sample, more typically ≤2 ppb, compared with <<2 ppb for preimplantation levels, well below reported toxic levels. Conclusions There was no evidence of adverse or abnormal pathology in tissue surrounding antimicrobially processed pacemakers, or deleterious effect on basic pacing capabilities and parameters at 12 weeks. This proof of concept study provides evidence of basic biocompatibility and feasibility of applying this silver ion-based antimicrobial surface to a titanium pacemaker surface. PMID:28674615

  18. Dual-function 2-nitroimidazoles as hypoxic cell radiosensitizers and bioreductive cytotoxins: In vivo evaluation in KHT murine sarcomas

    Energy Technology Data Exchange (ETDEWEB)

    Cole, S.; Stratford, I.J.; Adams, G.E.; Fielden, E.M.; Jenkins, T.C. (Medical Research Council, Didcot, Oxon (England))

    1990-10-01

    The efficacies of a series of potential prodrugs of RSU-1069 and its alkyl-aziridine analogues were assessed. These 1-(2-haloethylamino)-3-(2-nitro-1-imidazolyl)-2-propanol compounds were designed to cyclize in vivo to generate 2-nitro-imidazoles with aziridine (RSU-1069) or alkyl-substituted aziridine (RSU-1164, RB-7040, or RSU-1150) functions. Maximum tolerated single, intraperitoneal doses (MTD) were determined in C3H/He mice bearing subcutaneous KHT sarcomas, and a drug dose-response relationship for radiosensitization was established for each compound administered at the optimum time (45-60 min) before local irradiation of tumors with a 10-Gy dose of X-rays. The potentials of the compounds as bioreductive cytotoxins were studied by administering them immediately after irradiation. Tumor cell survival was measured 18-24 h after treatment in an in vitro soft agar clonogenic assay. Results of toxicity, radiosensitization, and bioreductive cytotoxicity assays for each of the prodrugs (RB-6171, RB-6172, RB-6173, RB-6174, and RB-6175) of the alkyl-substituted aziridines were entirely consistent with complete conversion to their respective target compounds. For example, RB-6171 (the prodrug form of RSU-1164) was only about four times less efficient than RSU-1069 as a radiosensitizer and bioreductive cytotoxin but had an MTD 7.5 times higher. In contrast, prodrugs of RSU-1069 (RB-6144 and RB-6145) were two- to threefold less toxic than their expected product. RB-6144 was a poor radiosensitizer and bioreductive agent compared with RSU-1069 and was similar to RB-6170, a nonalkylating nitroimidazole. This is consistent with the observation that there is limited conversion of RB-6144 to RSU-1069 in vitro. However, radiosensitization and bioreductive cytotoxicity produced by RB-6145 were only slightly less than the effects produced by RSU-1069.

  19. Angiotensin II impairs endothelial progenitor cell number and function in vitro and in vivo: implications for vascular regeneration.

    Science.gov (United States)

    Endtmann, Cathleen; Ebrahimian, Talin; Czech, Thomas; Arfa, Omar; Laufs, Ulrich; Fritz, Mathias; Wassmann, Kerstin; Werner, Nikos; Petoumenos, Vasileios; Nickenig, Georg; Wassmann, Sven

    2011-09-01

    vitro and in vivo, resulting in diminished vascular regeneration.

  20. In Vivo Models of Muscle Angiogenesis.

    Science.gov (United States)

    Egginton, Stuart

    2016-01-01

    Angiogenesis is an important determinant of tissue function, from delivery of oxygen and other substrates to removal of waste products, in health and disease (e.g., adaptive or pathological remodelling). The phenotype and functional responses of endothelial cells are conditioned by systemic humoral signals and local environmental factors, including the haemodynamic forces that act upon them. Here we describe some interventions that have been helpful in unraveling the integrative nature of the complex in vivo response, and quantitative assessment of angiogenesis in muscle.

  1. Human telomerase reverse transcriptase (hTERT Q169 is essential for telomerase function in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Haley D M Wyatt

    Full Text Available BACKGROUND: Telomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT, and an RNA subunit, the telomerase RNA (TR. Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: To understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT Q169 to create neutral (Q169A, conservative (Q169N, and non-conservative (Q169D mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function. CONCLUSIONS/SIGNIFICANCE: We provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme

  2. Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia

    Directory of Open Access Journals (Sweden)

    Serena Joaquín

    2009-06-01

    Full Text Available Abstract Background Studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. The normalization of relative qRT-PCR data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used. The present study aims to determine the expression stability of ten housekeeping genes (Gapdh, β2m, Hprt, Ppia, Rpl13a, Oaz1, 18S rRNA, Gusb, Ywhaz and Sdha to establish their suitability as control genes for in-vitro and in-vivo cerebral ischaemia models. Results The expression stability of the candidate reference genes was evaluated using the 2-ΔC'T method and ANOVA followed by Dunnett's test. For the in-vitro model using primary cultures of rat astrocytes, all genes analysed except for Rpl13a and Sdha were found to have significantly different levels of mRNA expression. These different levels were also found in the case of the in-vivo model of pMCAO in rats except for Hprt, Sdha and Ywhaz mRNA, where the expression did not vary. Sdha and Ywhaz were identified by geNorm and NormFinder as the two most stable genes. Conclusion We have validated endogenous control genes for qRT-PCR analysis of gene expression in in-vitro and in-vivo cerebral ischaemia models. For normalization purposes, Rpl13a and Sdha are found to be the most suitable genes for the in-vitro model and Sdha and Ywhaz for the in-vivo model. Genes previously used as housekeeping genes for the in-vivo model in the literature were not validated as good control genes in the present study, showing the need for careful evaluation for each new experimental setup.

  3. Quantitative Analysis of the Effective Functional Structure in Yeast Glycolysis

    Science.gov (United States)

    De la Fuente, Ildefonso M.; Cortes, Jesus M.

    2012-01-01

    The understanding of the effective functionality that governs the enzymatic self-organized processes in cellular conditions is a crucial topic in the post-genomic era. In recent studies, Transfer Entropy has been proposed as a rigorous, robust and self-consistent method for the causal quantification of the functional information flow among nonlinear processes. Here, in order to quantify the functional connectivity for the glycolytic enzymes in dissipative conditions we have analyzed different catalytic patterns using the technique of Transfer Entropy. The data were obtained by means of a yeast glycolytic model formed by three delay differential equations where the enzymatic rate equations of the irreversible stages have been explicitly considered. These enzymatic activity functions were previously modeled and tested experimentally by other different groups. The results show the emergence of a new kind of dynamical functional structure, characterized by changing connectivity flows and a metabolic invariant that constrains the activity of the irreversible enzymes. In addition to the classical topological structure characterized by the specific location of enzymes, substrates, products and feedback-regulatory metabolites, an effective functional structure emerges in the modeled glycolytic system, which is dynamical and characterized by notable variations of the functional interactions. The dynamical structure also exhibits a metabolic invariant which constrains the functional attributes of the enzymes. Finally, in accordance with the classical biochemical studies, our numerical analysis reveals in a quantitative manner that the enzyme phosphofructokinase is the key-core of the metabolic system, behaving for all conditions as the main source of the effective causal flows in yeast glycolysis. PMID:22393350

  4. Biocompatible photocrosslinked poly(ester anhydride) based on functionalized poly(epsilon-caprolactone) prepolymer shows surface erosion controlled drug release in vitro and in vivo.

    Science.gov (United States)

    Mönkäre, J; Hakala, R A; Vlasova, M A; Huotari, A; Kilpeläinen, M; Kiviniemi, A; Meretoja, V; Herzig, K H; Korhonen, H; Seppälä, J V; Järvinen, K

    2010-09-15

    Star-shaped poly(epsilon-caprolactone) oligomers functionalized with succinic anhydride were used as prepolymers to prepare photocrosslinked poly(ester anhydride) to evaluate their in vivo drug delivery functionality and biocompatibility. Thus, in this work, erosion, drug release and safety of the photocrosslinked poly(ester anhydride) were examined in vitro and in vivo. A small water-soluble drug, propranolol HCl (M(w) 296 g/mol, solubility 50 mg/ml), was used as the model drug in an evaluation of the erosion controlled release. Drug-free and drug-loaded (10-60% w/w) poly(ester anhydride) discoids eroded in vitro (pH 7.4 buffer, +37 degrees C) linearly within 24-48 h. A strong correlation between the polymer erosion and the linear drug release in vitro was observed, indicating that the release had been controlled by the erosion of the polymer. Similarly, in vivo studies (s.c. implantation of discoids in rats) indicated that surface erosion controlled drug release from the discoids (drug loading 40% w/w). Oligomers did not decrease cell viability in vitro and the implanted discoids (s.c., rats) did not evoke any cytokine activity in vivo. In summary, surface erosion controlled drug release and the safety of photocrosslinked poly(ester anhydride) were demonstrated in this study.

  5. A method to study in vivo stability of DNA nanostructures.

    Science.gov (United States)

    Surana, Sunaina; Bhatia, Dhiraj; Krishnan, Yamuna

    2013-11-01

    DNA nanostructures are rationally designed, synthetic, nanoscale assemblies obtained from one or more DNA sequences by their self-assembly. Due to the molecularly programmable as well as modular nature of DNA, such designer DNA architectures have great potential for in cellulo and in vivo applications. However, demonstrations of functionality in living systems necessitates a method to assess the in vivo stability of the relevant nanostructures. Here, we outline a method to quantitatively assay the stability and lifetime of various DNA nanostructures in vivo. This exploits the property of intact DNA nanostructures being uptaken by the coelomocytes of the multicellular model organism Caenorhabditis elegans. These studies reveal that the present fluorescence based assay in coelomocytes of C. elegans is an useful in vivo test bed for measuring DNA nanostructure stability.

  6. Isolation of human umbilical cord blood aldehyde dehydrogenase-expressing progenitor cells that modulate vascular regenerative functions in vitro and in vivo.

    Science.gov (United States)

    Putman, David M; Hess, David A

    2013-01-01

    This unit describes the isolation and application of human umbilical cord blood progenitor cells to modulate vascular regenerative functions using in vitro co-culture systems and in vivo transplantation models. Using aldehyde dehydrogenase as a marker of stem cell function, blood-derived progenitors can be efficiently purified form human umbilical cord blood using flow cytometry. We describe in vitro approaches to measure cell-mediated effects on the survival, proliferation, and tube-forming function of endothelial cells using growth-rate assays and Matrigel tube-forming assays. Additionally, we provide a detailed protocol for inducing acute unilateral hindlimb ischemia in immune-deficient mice to assess progenitor cell-modulated effects on vascular regeneration by tracking the recovery of blood flow using noninvasive laser Doppler perfusion imaging. Collectively, we present combined in vitro and in vivo transplantation strategies for the pre-clinical assessment of human progenitor cell-based therapies to treat ischemic disease.

  7. An Ultrasound Image-Based Dynamic Fusion Modeling Method for Predicting the Quantitative Impact of In Vivo Liver Motion on Intraoperative HIFU Therapies: Investigations in a Porcine Model.

    Directory of Open Access Journals (Sweden)

    W Apoutou N'Djin

    Full Text Available Organ motion is a key component in the treatment of abdominal tumors by High Intensity Focused Ultrasound (HIFU, since it may influence the safety, efficacy and treatment time. Here we report the development in a porcine model of an Ultrasound (US image-based dynamic fusion modeling method for predicting the effect of in vivo motion on intraoperative HIFU treatments performed in the liver in conjunction with surgery. A speckle tracking method was used on US images to quantify in vivo liver motions occurring intraoperatively during breathing and apnea. A fusion modeling of HIFU treatments was implemented by merging dynamic in vivo motion data in a numerical modeling of HIFU treatments. Two HIFU strategies were studied: a spherical focusing delivering 49 juxtapositions of 5-second HIFU exposures and a toroidal focusing using 1 single 40-second HIFU exposure. Liver motions during breathing were spatially homogenous and could be approximated to a rigid motion mainly encountered in the cranial-caudal direction (f = 0.20 Hz, magnitude > 13 mm. Elastic liver motions due to cardiovascular activity, although negligible, were detectable near millimeter-wide sus-hepatic veins (f = 0.96 Hz, magnitude 75%. Fusion modeling predictions were preliminarily validated in vivo and showed the potential of using a long-duration toroidal HIFU exposure to accelerate the ablation process during breathing (from 0.5 to 6 cm3 · min(-1. To improve HIFU treatment control, dynamic fusion modeling may be interesting for assessing numerically focusing strategies and motion compensation techniques in more realistic conditions.

  8. Ectodomains of the LDL receptor-related proteins LRP1b and LRP4 have anchorage independent functions in vivo.

    Directory of Open Access Journals (Sweden)

    Martin F Dietrich

    Full Text Available BACKGROUND: The low-density lipoprotein (LDL receptor gene family is a highly conserved group of membrane receptors with diverse functions in developmental processes, lipoprotein trafficking, and cell signaling. The low-density lipoprotein (LDL receptor-related protein 1b (LRP1B was reported to be deleted in several types of human malignancies, including non-small cell lung cancer. Our group has previously reported that a distal extracellular truncation of murine Lrp1b that is predicted to secrete the entire intact extracellular domain (ECD is fully viable with no apparent phenotype. METHODS AND PRINCIPAL FINDINGS: Here, we have used a gene targeting approach to create two mouse lines carrying internally rearranged exons of Lrp1b that are predicted to truncate the protein closer to the N-terminus and to prevent normal trafficking through the secretary pathway. Both mutations result in early embryonic lethality, but, as expected from the restricted expression pattern of LRP1b in vivo, loss of Lrp1b does not cause cellular lethality as homozygous Lrp1b-deficient blastocysts can be propagated normally in culture. This is similar to findings for another LDL receptor family member, Lrp4. We provide in vitro evidence that Lrp4 undergoes regulated intramembraneous processing through metalloproteases and gamma-secretase cleavage. We further demonstrate negative regulation of the Wnt signaling pathway by the soluble extracellular domain. CONCLUSIONS AND SIGNIFICANCE: Our results underline a crucial role for Lrp1b in development. The expression in mice of truncated alleles of Lrp1b and Lrp4 with deletions of the transmembrane and intracellular domains leads to release of the extracellular domain into the extracellular space, which is sufficient to confer viability. In contrast, null mutations are embryonically (Lrp1b or perinatally (Lrp4 lethal. These findings suggest that the extracellular domains of both proteins may function as a scavenger for

  9. Pulmonary alveolar proteinosis: Quantitative CT and pulmonary functional correlations

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Yubao, E-mail: yubaoguan@163.com [Department of Radiology, the First Affiliated Hospital of Guangzhou Medical College, Guangzhou 510120 (China); State Key Laboratory of Respiratory Disease, Guangzhou 510120 (China); Zeng, Qingsi [Department of Radiology, the First Affiliated Hospital of Guangzhou Medical College, Guangzhou 510120 (China); Yang, Haihong; Zheng, Jinping; Li, Shiyue; Gao, Yi [State Key Laboratory of Respiratory Disease, Guangzhou 510120 (China); Deng, Yu [Department of Radiology, the First Affiliated Hospital of Guangzhou Medical College, Guangzhou 510120 (China); Mei, Jiang [State Key Laboratory of Respiratory Disease, Guangzhou 510120 (China); He, Jianxing, E-mail: jianxing63@163.com [State Key Laboratory of Respiratory Disease, Guangzhou 510120 (China); Zhong, Nanshan, E-mail: nanshan@vip.163.com [State Key Laboratory of Respiratory Disease, Guangzhou 510120 (China)

    2012-09-15

    Objective: We assessed the relationship between quantitative computer tomography (qCT) and the pulmonary function test (PFT) or blood gas analysis in pulmonary alveolar proteinosis (PAP) patients, as well as the utility of these analyses to monitor responses to whole lung lavage (WLL) therapy. Methods: Thirty-eight PAP patients simultaneously received a CT scan and PFT. Fifteen of these patients, undergoing sequential WLL for a total of 20 lavages, also underwent chest CT scans and blood gas analysis before and after WLL, and 14 of 15 patients underwent simultaneous PFT analysis. Differences between the qCT and PFT results were analyzed by canonical correlation. Results: PAP patients with low predicted values for FVC, FEV1, D{sub LCO} and D{sub LCO}/VA indicated small airspace volume and mean lung inflation, low airspace volume/total lung volume ratio and high mean lung density. Correlation and regression analysis revealed a strong correlation between D{sub LCO} and PaO{sub 2} values with CT results. The qCT results indicated that WLL significantly decreased lung weights and mean lung densities, and improved the total airspace volume/total lung volume ratios and mean lung inflations. Conclusion: Quantitative CT may be a sensitive tool for measuring the response of PAP patients to medical interventions such as WLL.

  10. Regional homogeneity changes in hemodialysis patients with end stage renal disease: in vivo resting-state functional MRI study.

    Directory of Open Access Journals (Sweden)

    Cheng Li

    Full Text Available OBJECTIVE: To prospectively investigate and detect early cerebral regional homogeneity (ReHo changes in neurologically asymptomatic patients with end stage renal disease (ESRD using in vivo resting-state functional MR imaging (Rs-fMRI. METHODS: We enrolled 20 patients (15 men, 5 women; meanage, 37.1 years; range, 19-49 years with ESRD and 20 healthy controls (15 men, 5 women; mean age, 38.3 years; range, 28-49 years. The mean duration of hemodialysis for the patient group was 10.7±6.4 monthes. There was no significant sex or age difference between the ESRD and control groups. Rs-fMRI was performed using a gradient-echo echo-planar imaging sequence. ReHo was calculated using software (DPARSF. Voxel-based analysis of the ReHo maps between ESRD and control groups was performed with a two-samples t test. Statistical maps were set at P value less than 0.05 and were corrected for multiple comparisons. The Mini-Mental State Examination (MMSE was administered to all participants at imaging. RESULTS: ReHo values were increased in the bilateral superior temporal gyrus and left medial frontal gyrus in the ERSD group compared with controls, but a significantly decreased ReHo value was found in the right middle temporal gyrus. There was no significant correlation between ReHo values and the duration of hemodialysis in the ESRD group. Both the patients and control subjects had normal MMSE scores (≥28. CONCLUSIONS: Our finding revealed that abnormal brain activity was distributed mainly in the memory and cognition related cotices in patients with ESRD. The abnormal spontaneous neuronal activity in those areas provide information on the neural mechanisms underlying cognitive impairment in patients with ESRD, and demonstrate that Rs-fMRI with ReHo analysis is a useful non-invasive imaging tool for the detection of early cerebral ReHo changes in hemodialysis patients with ESRD.

  11. Analysis of the Peroxidase Activity of Rice (Oryza Sativa) Recombinant Hemoglobin 1: Implications for the In Vivo Function of Hexacoordinate Non-Symbiotic Hemoglobins in Plants

    Science.gov (United States)

    In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about the peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H2O2 system at pH ...

  12. Improved In Vitro and In Vivo Biocompatibility of Graphene Oxide through Surface Modification: Poly(Acrylic Acid)-Functionalization is Superior to PEGylation.

    Science.gov (United States)

    Xu, Ming; Zhu, Jianqiang; Wang, Fanfan; Xiong, Yunjing; Wu, Yakun; Wang, Qiuquan; Weng, Jian; Zhang, Zhihong; Chen, Wei; Liu, Sijin

    2016-03-22

    The unique physicochemical properties of two-dimensional (2D) graphene oxide (GO) could greatly benefit the biomedical field; however, recent research demonstrated that GO could induce in vitro and in vivo toxicity. We determined the mechanism of GO induced toxicity, and our in vitro experiments revealed that pristine GO could impair cell membrane integrity and functions including regulation of membrane- and cytoskeleton-associated genes, membrane permeability, fluidity and ion channels. Furthermore, GO induced platelet depletion, pro-inflammatory response and pathological changes of lung and liver in mice. To improve the biocompatibility of pristine GO, we prepared a series of GO derivatives including aminated GO (GO-NH2), poly(acrylamide)-functionalized GO (GO-PAM), poly(acrylic acid)-functionalized GO (GO-PAA) and poly(ethylene glycol)-functionalized GO (GO-PEG), and compared their toxicity with pristine GO in vitro and in vivo. Among these GO derivatives, GO-PEG and GO-PAA induced less toxicity than pristine GO, and GO-PAA was the most biocompatible one in vitro and in vivo. The differences in biocompatibility were due to the differential compositions of protein corona, especially immunoglobulin G (IgG), formed on their surfaces that determine their cell membrane interaction and cellular uptake, the extent of platelet depletion in blood, thrombus formation under short-term exposure and the pro-inflammatory effects under long-term exposure. Overall, our combined data delineated the key molecular mechanisms underlying the in vivo and in vitro biological behaviors and toxicity of pristine GO, and identified a safer GO derivative that could be used for future applications.

  13. Mechanism of Carbamate Inactivation of FAAH: Implications for the Design of Covalent Inhibitors and In Vivo Functional Probes for Enzymes

    Science.gov (United States)

    Alexander, Jessica P.; Cravatt, Benjamin F.

    2006-01-01

    Summary Fatty acid amide hydrolase (FAAH) regulates a large class of signaling lipids, including the endocannabinoid anandamide. Carbamate inhibitors of FAAH display analgesic and anxiolytic properties in rodents. However, the mechanism by which carbamates inhibit FAAH remains obscure. Here, we provide biochemical evidence that carbamates covalently modify the active site of FAAH by adopting an orientation opposite of that originally predicted from modeling. Based on these results, a series of carbamates was designed that display enhanced potency. One agent was converted into a “click chemistry” probe to comprehensively evaluate the proteome reactivity of FAAH-directed carbamates in vivo. These inhibitors were selective for FAAH in the nervous system, but they reacted with several enzymes in peripheral tissues. The experimental strategy described herein can be used to create in vivo probes for any enzyme susceptible to covalent inhibition. PMID:16298297

  14. Effects of Acer okamotoanum sap on the function of polymorphonuclear neutrophilic leukocytes in vitro and in vivo.

    Science.gov (United States)

    An, Beum-Soo; Kang, Ji-Houn; Yang, Hyun; Yang, Mhan-Pyo; Jeung, Eui-Bae

    2013-02-01

    Sap is a plant fluid that primarily consists of water and small amounts of mineral elements, sugars, hormones and other nutrients. Acer mono (A. mono) is an endemic Korean mono maple which was recently suggested to have health benefits due to its abundant calcium and magnesium ion content. In the present study, we examined the effects of sap from Acer okamotoanum (A. okamotoanum) on the phagocytic response of mouse neutrophils in vivo and rat and canine neutrophils in vitro. We tested the regulation of phagocytic activity, oxidative burst activity (OBA) and the levels of filamentous polymeric actin (F-actin) in the absence and presence of dexamethasone (DEX) in vitro and in vivo. Our results showed that DEX primarily reduced OBA in the mouse neutrophils, and that this was reversed in the presence of the sap. By contrast, the phagocytic activity of the mouse cells was not regulated by either DEX or the sap. Rat and canine polymorphonuclear neutrophilic leukocytes (PMNs) responded in vitro to the sap in a similar manner by increasing OBA. However, regulation of phagocytic activity by the sap was different between the species. In canine PMNs, phagocytic activity was enhanced by the sap at a high dose, while it did not significantly modulate this activity in rat PMNs. These findings suggest that the sap of A. okamotoanum stimulates neutrophil activity in the mouse, rat and canine by increasing OBA in vivo and in vitro, and thus may have a potential antimicrobial effect in the PMNs of patients with infections.

  15. Functional consequences of T-stem mutations in E. coli tRNA Thr UGU in vitro and in vivo

    DEFF Research Database (Denmark)

    Saks, Margaret E; Sanderson, Lee E; Choi, Daniel S;

    2011-01-01

    unable to support growth of E. coli or are less effective than the wild-type sequence. Since the inviable T-stem sequences are often present in other E. coli tRNAs, it appears that T-stem sequences in each tRNA body have evolved to optimize function in a different way. Although mutations of tRNAThr can...... to mutations in three T-stem base pairs in a quantitatively identical manner. However, tRNAThr differs from other tRNAs by also using its rare A52–C62 pair as a negative specificity determinant. Using a plasmid-based tRNA gene replacement strategy, we show that many of the tRNAThrUGU T-stem changes are either......The binding affinities between Escherichia coli EF-Tu and 34 single and double base-pair changes in the T stem of E. coli tRNAThrUGU were compared with similar data obtained previously for several aa-tRNAs binding to Thermus thermophilus EF-Tu. With a single exception, the two proteins bound...

  16. Epistasis analysis for quantitative traits by functional regression model.

    Science.gov (United States)

    Zhang, Futao; Boerwinkle, Eric; Xiong, Momiao

    2014-06-01

    The critical barrier in interaction analysis for rare variants is that most traditional statistical methods for testing interactions were originally designed for testing the interaction between common variants and are difficult to apply to rare variants because of their prohibitive computational time and poor ability. The great challenges for successful detection of interactions with next-generation sequencing (NGS) data are (1) lack of methods for interaction analysis with rare variants, (2) severe multiple testing, and (3) time-consuming computations. To meet these challenges, we shift the paradigm of interaction analysis between two loci to interaction analysis between two sets of loci or genomic regions and collectively test interactions between all possible pairs of SNPs within two genomic regions. In other words, we take a genome region as a basic unit of interaction analysis and use high-dimensional data reduction and functional data analysis techniques to develop a novel functional regression model to collectively test interactions between all possible pairs of single nucleotide polymorphisms (SNPs) within two genome regions. By intensive simulations, we demonstrate that the functional regression models for interaction analysis of the quantitative trait have the correct type 1 error rates and a much better ability to detect interactions than the current pairwise interaction analysis. The proposed method was applied to exome sequence data from the NHLBI's Exome Sequencing Project (ESP) and CHARGE-S study. We discovered 27 pairs of genes showing significant interactions after applying the Bonferroni correction (P-values < 4.58 × 10(-10)) in the ESP, and 11 were replicated in the CHARGE-S study.

  17. A high-affinity interaction with ADP-actin monomers underlies the mechanism and in vivo function of Srv2/cyclase-associated protein.

    Science.gov (United States)

    Mattila, Pieta K; Quintero-Monzon, Omar; Kugler, Jamie; Moseley, James B; Almo, Steven C; Lappalainen, Pekka; Goode, Bruce L

    2004-11-01

    Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 microM) compared with ATP-G-actin (Kd 1.9 microM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved beta-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly.

  18. Construction of an in vivo nonsense readthrough assay system and functional analysis of ribosomal proteins S12, S4, and S5 in Bacillus subtilis.

    Science.gov (United States)

    Inaoka, T; Kasai, K; Ochi, K

    2001-09-01

    To investigate the function of ribosomal proteins and translational factors in Bacillus subtilis, we developed an in vivo assay system to measure the level of nonsense readthrough by utilizing the LacZ-LacI system. Using the in vivo nonsense readthrough assay system which we developed, together with an in vitro poly(U)-directed cell-free translation assay system, we compared the processibility and translational accuracy of mutant ribosomes with those of the wild-type ribosome. Like Escherichia coli mutants, most S12 mutants exhibited lower frequencies of both UGA readthrough and missense error; the only exception was a mutant (in which Lys-56 was changed to Arg) which exhibited a threefold-higher frequency of readthrough than the wild-type strain. We also isolated several ribosomal ambiguity (ram) mutants from an S12 mutant. These ram mutants and the S12 mutant mentioned above (in which Lys-56 was changed to Arg) exhibited higher UGA readthrough levels. Thus, the mutation which altered Lys-56 to Arg resulted in a ram phenotype in B. subtilis. The efficacy of our in vivo nonsense readthrough assay system was demonstrated in our investigation of the function of ribosomal proteins and translational factors.

  19. In Vivo Determination of Mitochondrial Function Using Luciferase-Expressing Caenorhabditis elegans: Contribution of Oxidative Phosphorylation, Glycolysis, and Fatty Acid Oxidation to Toxicant-Induced Dysfunction.

    Science.gov (United States)

    Luz, Anthony L; Lagido, Cristina; Hirschey, Matthew D; Meyer, Joel N

    2016-08-01

    Mitochondria are a target of many drugs and environmental toxicants; however, how toxicant-induced mitochondrial dysfunction contributes to the progression of human disease remains poorly understood. To address this issue, in vivo assays capable of rapidly assessing mitochondrial function need to be developed. Here, using the model organism Caenorhabditis elegans, we describe how to rapidly assess the in vivo role of the electron transport chain, glycolysis, or fatty acid oxidation in energy metabolism following toxicant exposure, using a luciferase-expressing ATP reporter strain. Alterations in mitochondrial function subsequent to toxicant exposure are detected by depleting steady-state ATP levels with inhibitors of the mitochondrial electron transport chain, glycolysis, or fatty acid oxidation. Differential changes in ATP following short-term inhibitor exposure indicate toxicant-induced alterations at the site of inhibition. Because a microplate reader is the only major piece of equipment required, this is a highly accessible method for studying toxicant-induced mitochondrial dysfunction in vivo. © 2016 by John Wiley & Sons, Inc.

  20. In vitro and non-invasive in vivo effects of the cannabinoid-1 receptor (CB1R) agonist AM841 on gastrointestinal motor function in the rat

    Science.gov (United States)

    Abalo, R; Chen, C; Vera, G; Fichna, J; Thakur, GA; López-Pérez, AE; Makriyannis, A; Martín-Fontelles, MI; Storr, M

    2015-01-01

    Background Cannabinoids have been traditionally used for the treatment of gastrointestinal (GI) symptoms, but the associated central effects, through cannabinoid-1 receptors (CB1R), constitute an important drawback. Our aims were to characterize the effects of the recently developed highly potent long-acting megagonist AM841 on GI motor function and to determine its central effects in rats. Methods Male Wistar rats were used for in vitro and in vivo studies. The effect of AM841 was tested on electrically-induced twitch contractions of GI preparations (in vitro) and on GI motility measured radiographically after contrast administration (in vivo). Central effects of AM841 were evaluated using the cannabinoid tetrad. The non-selective cannabinoid agonist WIN 55,212-2 (WIN) was used for comparison. The CB1R (AM251) and CB2R (AM630) antagonists were used to characterize cannabinoid receptor-mediated effects of AM841. Key results AM841 dose-dependently reduced in vitro contractile activity of rat GI preparations via CB1R, but not CB2R or opioid receptors. In vivo, AM841 acutely and potently reduced gastric emptying and intestinal transit in a dose-dependent and AM251-sensitive manner. The in vivo GI effects of AM841 at 0.1 mg kg−1 were comparable to those induced by WIN at 5 mg kg−1. However, at this dose, AM841 did not induce any sign of the cannabinoid tetrad, whereas WIN induced significant central effects. Conclusions & Inferences The CB1R megagonist AM841 may potently depress GI motor function in the absence of central effects. This effect may be mediated peripherally and may be useful in the treatment of GI motility disorders. PMID:26387676

  1. Quantitative Functional Imaging Using Dynamic Positron Computed Tomography and Rapid Parameter Estimation Techniques

    Science.gov (United States)

    Koeppe, Robert Allen

    Positron computed tomography (PCT) is a diagnostic imaging technique that provides both three dimensional imaging capability and quantitative measurements of local tissue radioactivity concentrations in vivo. This allows the development of non-invasive methods that employ the principles of tracer kinetics for determining physiological properties such as mass specific blood flow, tissue pH, and rates of substrate transport or utilization. A physiologically based, two-compartment tracer kinetic model was derived to mathematically describe the exchange of a radioindicator between blood and tissue. The model was adapted for use with dynamic sequences of data acquired with a positron tomograph. Rapid estimation techniques were implemented to produce functional images of the model parameters by analyzing each individual pixel sequence of the image data. A detailed analysis of the performance characteristics of three different parameter estimation schemes was performed. The analysis included examination of errors caused by statistical uncertainties in the measured data, errors in the timing of the data, and errors caused by violation of various assumptions of the tracer kinetic model. Two specific radioindicators were investigated. ('18)F -fluoromethane, an inert freely diffusible gas, was used for local quantitative determinations of both cerebral blood flow and tissue:blood partition coefficient. A method was developed that did not require direct sampling of arterial blood for the absolute scaling of flow values. The arterial input concentration time course was obtained by assuming that the alveolar or end-tidal expired breath radioactivity concentration is proportional to the arterial blood concentration. The scale of the input function was obtained from a series of venous blood concentration measurements. The method of absolute scaling using venous samples was validated in four studies, performed on normal volunteers, in which directly measured arterial concentrations

  2. Cathelicidin host defence peptide augments clearance of pulmonary Pseudomonas aeruginosa infection by its influence on neutrophil function in vivo.

    Directory of Open Access Journals (Sweden)

    Paula E Beaumont

    Full Text Available Cathelicidins are multifunctional cationic host-defence peptides (CHDP; also known as antimicrobial peptides and an important component of innate host defence against infection. In addition to microbicidal potential, these peptides have properties with the capacity to modulate inflammation and immunity. However, the extent to which such properties play a significant role during infection in vivo has remained unclear. A murine model of acute P. aeruginosa lung infection was utilised, demonstrating cathelicidin-mediated enhancement of bacterial clearance in vivo. The delivery of exogenous synthetic human cathelicidin LL-37 was found to enhance a protective pro-inflammatory response to infection, effectively promoting bacterial clearance from the lung in the absence of direct microbicidal activity, with an enhanced early neutrophil response that required both infection and peptide exposure and was independent of native cathelicidin production. Furthermore, although cathelicidin-deficient mice had an intact early cellular inflammatory response, later phase neutrophil response to infection was absent in these animals, with significantly impaired clearance of P. aeruginosa. These findings demonstrate the importance of the modulatory properties of cathelicidins in pulmonary infection in vivo and highlight a key role for cathelicidins in the induction of protective pulmonary neutrophil responses, specific to the infectious milieu. In additional to their physiological roles, CHDP have been proposed as future antimicrobial therapeutics. Elucidating and utilising the modulatory properties of cathelicidins has the potential to inform the development of synthetic peptide analogues and novel therapeutic approaches based on enhancing innate host defence against infection with or without direct microbicidal targeting of pathogens.

  3. Data in support of in vivo studies of silk based gold nano-composite conduits for functional peripheral nerve regeneration

    Directory of Open Access Journals (Sweden)

    Suradip Das

    2015-09-01

    Full Text Available In the present data article we report the in vitro and in vivo biocompatibility of fabricated nerve conduits described in Das et al. [1]. Green synthesised gold nanoparticles (GNPs were evaluated for their cytotoxicity in rat Schwann cells (SCTM41. We also describe herein the adhesion and proliferation of Schwann cells over the nanofibrous scaffolds. Methods describing surgical implantation of conduits in a rat sciatic nerve injury model, confirming its accurate implantation as well as the porosity and swelling tendency of the nerve conduits are illustrated in the various figures and graphs.

  4. Tyrosine 129 of the murine gammaherpesvirus M2 protein is critical for M2 function in vivo.

    Directory of Open Access Journals (Sweden)

    Udaya S Rangaswamy

    Full Text Available A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes--predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68 infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe in vitro and in vivo studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells in vitro, only Y129 is critical for reactivation from latency and plasma cell differentiation in vivo.

  5. Tyrosine 129 of the murine gammaherpesvirus M2 protein is critical for M2 function in vivo.

    Science.gov (United States)

    Rangaswamy, Udaya S; O'Flaherty, Brigid M; Speck, Samuel H

    2014-01-01

    A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes--predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68) infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe in vitro and in vivo studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells in vitro, only Y129 is critical for reactivation from latency and plasma cell differentiation in vivo.

  6. A Salmonella Typhimurium-Typhi genomic chimera: a model to study Vi polysaccharide capsule function in vivo.

    Directory of Open Access Journals (Sweden)

    Angela M Jansen

    2011-07-01

    Full Text Available The Vi capsular polysaccharide is a virulence-associated factor expressed by Salmonella enterica serotype Typhi but absent from virtually all other Salmonella serotypes. In order to study this determinant in vivo, we characterised a Vi-positive S. Typhimurium (C5.507 Vi(+, harbouring the Salmonella pathogenicity island (SPI-7, which encodes the Vi locus. S. Typhimurium C5.507 Vi(+ colonised and persisted in mice at similar levels compared to the parent strain, S. Typhimurium C5. However, the innate immune response to infection with C5.507 Vi(+ and SGB1, an isogenic derivative not expressing Vi, differed markedly. Infection with C5.507 Vi(+ resulted in a significant reduction in cellular trafficking of innate immune cells, including PMN and NK cells, compared to SGB1 Vi(- infected animals. C5.507 Vi(+ infection stimulated reduced numbers of TNF-α, MIP-2 and perforin producing cells compared to SGB1 Vi(-. The modulating effect associated with Vi was not observed in MyD88(-/- and was reduced in TLR4(-/- mice. The presence of the Vi capsule also correlated with induction of the anti-inflammatory cytokine IL-10 in vivo, a factor that impacted on chemotaxis and the activation of immune cells in vitro.

  7. In vivo functional connectome of human brainstem nuclei of the ascending arousal, autonomic and motor systems by high spatial resolution 7 Tesla fMRI

    Science.gov (United States)

    Bianciardi, Marta; Toschi, Nicola; Eichner, Cornelius; Polimeni, Jonathan R.; Setsompop, Kawin; Brown, Emery N.; Hamalainen, Matti S.; Rosen, Bruce R.; Wald, Lawrence L.

    2016-01-01

    Object To map the in vivo human functional connectivity of several brainstem nuclei with the rest of the brain by using seed-based correlation of ultra-high magnetic field functional magnetic resonance imaging (fMRI) data. Materials and Methods We used the recently developed template of 11 brainstem nuclei derived from multi-contrast structural MRI at 7 Tesla as seed regions to determine their connectivity to the rest of the brain. To achieve this, we utilized the increased contrast-to-noise ratio of 7 Tesla fMRI compared to 3 Tesla and the time efficient simultaneous multi-slice imaging to cover the brain with high spatial resolution (1.1 mm-isotropic nominal resolution) while maintaining a short repetition time (2.5 s). Results The delineated Pearson’s correlation-based functional connectivity diagrams (connectomes) of 11 brainstem nuclei of the ascending arousal, motor and autonomic systems from 12 controls are presented and discussed in the context of existing histology and animal work. Conclusion Considering that the investigated brainstem nuclei play a crucial role in several vital functions, the delineated preliminary connectomes might prove useful for future in vivo research and clinical studies of human brainstem function and pathology, including disorders of consciousness, sleep disorders, autonomic disorders, Parkinson’s disease and other motor disorders. PMID:27126248

  8. In vivo functional connectome of human brainstem nuclei of the ascending arousal, autonomic, and motor systems by high spatial resolution 7-Tesla fMRI.

    Science.gov (United States)

    Bianciardi, Marta; Toschi, Nicola; Eichner, Cornelius; Polimeni, Jonathan R; Setsompop, Kawin; Brown, Emery N; Hämäläinen, Matti S; Rosen, Bruce R; Wald, Lawrence L

    2016-06-01

    Our aim was to map the in vivo human functional connectivity of several brainstem nuclei with the rest of the brain by using seed-based correlation of ultra-high magnetic field functional magnetic resonance imaging (fMRI) data. We used the recently developed template of 11 brainstem nuclei derived from multi-contrast structural MRI at 7 Tesla as seed regions to determine their connectivity to the rest of the brain. To achieve this, we used the increased contrast-to-noise ratio of 7-Tesla fMRI compared with 3 Tesla and time-efficient simultaneous multi-slice imaging to cover the brain with high spatial resolution (1.1-mm isotropic nominal resolution) while maintaining a short repetition time (2.5 s). The delineated Pearson's correlation-based functional connectivity diagrams (connectomes) of 11 brainstem nuclei of the ascending arousal, motor, and autonomic systems from 12 controls are presented and discussed in the context of existing histology and animal work. Considering that the investigated brainstem nuclei play a crucial role in several vital functions, the delineated preliminary connectomes might prove useful for future in vivo research and clinical studies of human brainstem function and pathology, including disorders of consciousness, sleep disorders, autonomic disorders, Parkinson's disease, and other motor disorders.

  9. 联合应用实时量RT-PCR和激光显微切割检测单个肝细胞RNA的表达%Quantitative analysis of RNA levels from single hepatocytes in vivo: combined use of real- time RT- PCR and laser microdissection

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: The manner in which a cell responds to and influences its environment is ultimately determined by the genes that are expressed. To better understand cellular functions, the isolation of single cells and subsequent quantification of the expressed genes is essential. METHODS: Normal liver tissue was obtained from operation, snapfrozen in liquid nitrogen and sectioned in crystat. Individual hepatocytes were microdissected. RNA was extracted, then reverse transcribed and amplified using real- time quantitative polymerase chain reaction (PCR). RESULTS: Single hepatocytes were dissected by laser beam and catapulted to the microcentrifuge cap which was put above the slide. In this way,cells were collected, RNA was extracted, reverse transcribed to cDNA and used for analysis of RNA expression by realtime quantitative PCR. The amplification results showed that quantitation of the RNA inside the cell was compatible with the number of cells. CONCLUSION: The expression of RNA in single cells can be quantitated successfully by using laser microdissection and real -time PCR. These techniques provide an opportunity to monitor in vivo gene expression levels in single hepatocytes.

  10. Quantitative assessment of hemodynamic and structural characteristics of in vivo brain tissue using total diffuse reflectance spectrum measured in a non-contact fashion.

    Science.gov (United States)

    Song, Yinchen; Garcia, Sarahy; Frometa, Yisel; Ramella-Roman, Jessica C; Soltani, Mohammad; Almadi, Mohamed; Riera, Jorge J; Lin, Wei-Chiang

    2017-01-01

    Here we present a new methodology that investigates the intrinsic structural and hemodynamic characteristics of in vivo brain tissue, in a non-contact fashion, and can be easily incorporated in an intra-operative environment. Within this methodology, relative total diffuse reflectance spectra (RTD(λ)) were acquired from targets using a hybrid spectroscopy imaging system. A spectral interpretation algorithm was subsequently applied to RTD(λ) to retrieve optical properties related to the compositional and structural characteristics of each target. Estimation errors of the proposed methodology were computationally evaluated using a Monte Carlo simulation model for photon migration under various conditions. It was discovered that this new methodology could handle moderate noise and achieve very high accuracy, but only if the refractive index of the target is known. The accuracy of the technique was also validated using a series of tissue phantom studies, and consistent and accurate estimates of μs'(λ)/μa(λ) were obtained from all the phantoms tested. Finally, a small-scale animal study was conducted to demonstrate the clinical utility of the reported method, wherein a forepaw stimulation model was utilized to induce transient hemodynamic responses in somatosensory cortices. With this approach, significant stimulation-related changes (p < 0.001) in cortical hemodynamic and structural characteristics were successfully measured.

  11. Quantitative in vivo cell-surface receptor imaging in oncology: kinetic modeling and paired-agent principles from nuclear medicine and optical imaging

    Science.gov (United States)

    Tichauer, Kenneth M.; Wang, Yu; Pogue, Brian W.; Liu, Jonathan T. C.

    2015-07-01

    The development of methods to accurately quantify cell-surface receptors in living tissues would have a seminal impact in oncology. For example, accurate measures of receptor density in vivo could enhance early detection or surgical resection of tumors via protein-based contrast, allowing removal of cancer with high phenotype specificity. Alternatively, accurate receptor expression estimation could be used as a biomarker to guide patient-specific clinical oncology targeting of the same molecular pathway. Unfortunately, conventional molecular contrast-based imaging approaches are not well adapted to accurately estimating the nanomolar-level cell-surface receptor concentrations in tumors, as most images are dominated by nonspecific sources of contrast such as high vascular permeability and lymphatic inhibition. This article reviews approaches for overcoming these limitations based upon tracer kinetic modeling and the use of emerging protocols to estimate binding potential and the related receptor concentration. Methods such as using single time point imaging or a reference-tissue approach tend to have low accuracy in tumors, whereas paired-agent methods or advanced kinetic analyses are more promising to eliminate the dominance of interstitial space in the signals. Nuclear medicine and optical molecular imaging are the primary modalities used, as they have the nanomolar level sensitivity needed to quantify cell-surface receptor concentrations present in tissue, although each likely has a different clinical niche.

  12. Pace of macrophage recruitment during different stages of soft tissue infection: Semi-quantitative evaluation by in vivo magnetic resonance imaging

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jin Seong; Sohn, Jin Young [University of Ulsan College of Medicine, Asan Medical Center, Laboratory for Molecular and Functional Imaging, Department of Radiology and Research Institute of Radiology, Seoul (Korea); Jung, Hyun-Don; Kim, Sang-Tae [University of Ulsan College of Medicine, Asan Institute for Life Sciences, Seoul (Korea); Lee, Kyoung Geun [Korea University College of Life Sciences and Biotechnology, Division of Biotechnology, Seoul (Korea); Kang, Hee Jung [Hallym University College of Medicine, Department of Laboratory Medicine, Anyang (Korea)

    2008-10-15

    We describe the pace of recruitment of iron-oxide-labeled macrophages to the site of different stages of infection by in vivo magnetic resonance (MR) imaging. Peritoneal macrophages were labeled with superparamagnetic iron oxide ex vivo and administered through the tail vein 6 (acute) or 48 (subacute) h after bacterial inoculation. The legs of the mice were imaged sequentially on a 4.7-T MR unit before and 3, 6, 12, 18, 24, 48 and 72 h after macrophage administration. The band-shaped lower signal intensity zone around the abscess on T2*-weighted GRE images became more obvious due to recruited macrophages up until 24 h after injection in the subacute and 48 h after injection in the acute group, indicating that the relative SI of the abscess wall decreased more rapidly and the pace of recruitment of macrophages was faster in the subacute than in the acute group. Chemokine antibody arrays of mouse sera detected increased concentration of granulocyte-colony-stimulating factor and tissue inhibitor of metalloproteinase-1 beginning at 12 h and increased interleukin-13 at 18 h. Monocyte chemoattractant protein-1 and macrophage-colony-stimulating factor began to increase at 96 h after infection. This difference in pace of recruitment may result from the release of chemokines. (orig.)

  13. Quantitative assessment of hemodynamic and structural characteristics of in vivo brain tissue using total diffuse reflectance spectrum measured in a non-contact fashion

    Science.gov (United States)

    Song, Yinchen; Garcia, Sarahy; Frometa, Yisel; Ramella-Roman, Jessica C.; Soltani, Mohammad; Almadi, Mohamed; Riera, Jorge J.; Lin, Wei-Chiang

    2016-01-01

    Here we present a new methodology that investigates the intrinsic structural and hemodynamic characteristics of in vivo brain tissue, in a non-contact fashion, and can be easily incorporated in an intra-operative environment. Within this methodology, relative total diffuse reflectance spectra (RTD(λ)) were acquired from targets using a hybrid spectroscopy imaging system. A spectral interpretation algorithm was subsequently applied to RTD(λ) to retrieve optical properties related to the compositional and structural characteristics of each target. Estimation errors of the proposed methodology were computationally evaluated using a Monte Carlo simulation model for photon migration under various conditions. It was discovered that this new methodology could handle moderate noise and achieve very high accuracy, but only if the refractive index of the target is known. The accuracy of the technique was also validated using a series of tissue phantom studies, and consistent and accurate estimates of μs’(λ)/μa(λ) were obtained from all the phantoms tested. Finally, a small-scale animal study was conducted to demonstrate the clinical utility of the reported method, wherein a forepaw stimulation model was utilized to induce transient hemodynamic responses in somatosensory cortices. With this approach, significant stimulation-related changes (p < 0.001) in cortical hemodynamic and structural characteristics were successfully measured. PMID:28101403

  14. In vivo functional imaging of blood flow and wall strain rate in outflow tract of embryonic chick heart using ultrafast spectral domain optical coherence tomography

    Science.gov (United States)

    Li, Peng; Yin, Xin; Shi, Liang; Rugonyi, Sandra; Wang, Ruikang K.

    2012-09-01

    During cardiac development, the cardiac wall and flowing blood are two important cardiac tissues that constantly interact with each other. This dynamic interaction defines appropriate biomechanical environment to which the embryonic heart is exposed. Quantitative assessment of the dynamic parameters of wall tissues and blood flow is required to further our understanding of cardiac development. We report the use of an ultrafast 1310-nm dual-camera spectral domain optical coherence tomography (SDOCT) system to characterize/image, in parallel, the dynamic radial strain rate of the myocardial wall and the Doppler velocity of the underlying flowing blood within an in vivo beating chick embryo. The OCT system operates at 184-kHz line scan rate, providing the flexibility of imaging the fast blood flow and the slow tissue deformation within one scan. The ability to simultaneously characterize tissue motion and blood flow provides a useful approach to better understand cardiac dynamics during early developmental stages.

  15. Establishment of a transgenic zebrafish line for superficial skin ablation and functional validation of apoptosis modulators in vivo.

    Directory of Open Access Journals (Sweden)

    Chi-Fang Chen

    Full Text Available BACKGROUND: Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR(cy17 (killer line, which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR(+ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR(+ signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR(+ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+ fluorescent signaling. CONCLUSION/SIGNIFICANCE: The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and

  16. Single-walled carbon nanotubes noncovalently functionalized with lipid modified polyethylenimine for siRNA delivery in vitro and in vivo.

    Science.gov (United States)

    Siu, King S; Zheng, Xiufen; Liu, Yanling; Zhang, Yujuan; Zhang, Xusheng; Chen, Di; Yuan, Ken; Gillies, Elizabeth R; Koropatnick, James; Min, Wei-Ping

    2014-10-15

    siRNA can downregulate the expression of specific genes. However, delivery to specific cells and tissues in vivo presents significant challenges. Modified carbon nanotubes (CNTs) have been shown to protect siRNA and facilitate its entry into cells. However, simple and efficient methods to functionalize CNTs are needed. Here, noncovalent functionalization of CNTs is performed and shown to effectively deliver siRNA to target cells. Specifically, single-walled CNTs were functionalized by noncovalent association with a lipopolymer. The lipopolymer (DSPE-PEG) was composed of a phospholipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and poly(ethylene glycol) (PEG). Three different ratios of polyethylenimine (PEI) to DSPE-PEG were synthesized and characterized and the products were used to disperse CNTs. The resulting materials were used for siRNA delivery in vitro and in vivo. The structural, biophysical, and biological properties of DGI/C and their complexes formed with siRNA were investigated. Cytotoxicity of the materials was low, and effective gene silencing in B16-F10 cells was demonstrated in vitro. In addition, significant uptake of siRNA as well as gene silencing in the liver was found following intravenous injection. This approach provides a new strategy for siRNA delivery and could provide insight for the development of noncovalently functionalized CNTs for siRNA therapy.

  17. ALA-PpIX variability quantitatively imaged in A431 epidermoid tumors using in vivo ultrasound fluorescence tomography and ex vivo assay

    Science.gov (United States)

    DSouza, Alisha V.; Flynn, Brendan P.; Gunn, Jason R.; Samkoe, Kimberley S.; Anand, Sanjay; Maytin, Edward V.; Hasan, Tayyaba; Pogue, Brian W.

    2014-03-01

    Treatment monitoring of Aminolevunilic-acid (ALA) - Photodynamic Therapy (PDT) of basal-cell carcinoma (BCC) calls for superficial and subsurface imaging techniques. While superficial imagers exist for this purpose, their ability to assess PpIX levels in thick lesions is poor; additionally few treatment centers have the capability to measure ALA-induced PpIX production. An area of active research is to improve treatments to deeper and nodular BCCs, because treatment is least effective in these. The goal of this work was to understand the logistics and technical capabilities to quantify PpIX at depths over 1mm, using a novel hybrid ultrasound-guided, fiber-based fluorescence molecular spectroscopictomography system. This system utilizes a 633nm excitation laser and detection using filtered spectrometers. Source and detection fibers are collinear so that their imaging plane matches that of ultrasound transducer. Validation with phantoms and tumor-simulating fluorescent inclusions in mice showed sensitivity to fluorophore concentrations as low as 0.025μg/ml at 4mm depth from surface, as presented in previous years. Image-guided quantification of ALA-induced PpIX production was completed in subcutaneous xenograft epidermoid cancer tumor model A431 in nude mice. A total of 32 animals were imaged in-vivo, using several time points, including pre-ALA, 4-hours post-ALA, and 24-hours post-ALA administration. On average, PpIX production in tumors increased by over 10-fold, 4-hours post-ALA. Statistical analysis of PpIX fluorescence showed significant difference among all groups; p<0.05. Results were validated by exvivo imaging of resected tumors. Details of imaging, analysis and results will be presented to illustrate variability and the potential for imaging these values at depth.

  18. A High-affinity Interaction with ADP-Actin Monomers Underlies the Mechanism and In Vivo Function of Srv2/cyclase-associated ProteinD⃞

    OpenAIRE

    Mattila, Pieta K.; Quintero-Monzon, Omar; Kugler, Jamie; Moseley, James B.; Almo, Steven C.; Lappalainen, Pekka; Goode, Bruce L

    2004-01-01

    Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 μM) compared with ATP-G-actin (Kd 1.9 μM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer additi...

  19. One-step synthesis of amino-functionalized ultrasmall near infrared-emitting persistent luminescent nanoparticles for in vitro and in vivo bioimaging

    Science.gov (United States)

    Shi, Junpeng; Sun, Xia; Zhu, Jianfei; Li, Jinlei; Zhang, Hongwu

    2016-05-01

    Near infrared (NIR)-emitting persistent luminescent nanoparticles (NPLNPs) have attracted much attention in bioimaging because they can provide long-term in vivo imaging with a high signal-to-noise ratio (SNR). However, conventional NPLNPs with large particle sizes that lack modifiable surface groups suffer from many serious limitations in bioimaging. Herein, we report a one-step synthesis of amino-functionalized ZnGa2O4:Cr,Eu nanoparticles (ZGO) that have an ultrasmall size, where ethylenediamine served as the reactant to fabricate the ZGO as well as the surfactant ligand to control the nanocrystal size and form surface amino groups. The ZGO exhibited a narrow particle size distribution, a bright NIR emission and a long afterglow luminescence. In addition, due to the excellent conjugation ability of the surface amino groups, the ZGO can be easily conjugated with many bio-functional molecules, which has been successfully utilized to realize in vitro and in vivo imaging. More importantly, the ZGO achieved re-excitation imaging using 650 nm and 808 nm NIR light in situ, which is advantageous for long-term and higher SNR bioimaging.Near infrared (NIR)-emitting persistent luminescent nanoparticles (NPLNPs) have attracted much attention in bioimaging because they can provide long-term in vivo imaging with a high signal-to-noise ratio (SNR). However, conventional NPLNPs with large particle sizes that lack modifiable surface groups suffer from many serious limitations in bioimaging. Herein, we report a one-step synthesis of amino-functionalized ZnGa2O4:Cr,Eu nanoparticles (ZGO) that have an ultrasmall size, where ethylenediamine served as the reactant to fabricate the ZGO as well as the surfactant ligand to control the nanocrystal size and form surface amino groups. The ZGO exhibited a narrow particle size distribution, a bright NIR emission and a long afterglow luminescence. In addition, due to the excellent conjugation ability of the surface amino groups, the ZGO can be

  20. Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo.

    Science.gov (United States)

    Dislich, Bastian; Wohlrab, Felix; Bachhuber, Teresa; Müller, Stephan A; Kuhn, Peer-Hendrik; Hogl, Sebastian; Meyer-Luehmann, Melanie; Lichtenthaler, Stefan F

    2015-10-01

    Analysis of murine cerebrospinal fluid (CSF) by quantitative mass spectrometry is challenging because of low CSF volume, low total protein concentration, and the presence of highly abundant proteins such as albumin. We demonstrate that the CSF proteome of individual mice can be analyzed in a quantitative manner to a depth of several hundred proteins in a robust and simple workflow consisting of single ultra HPLC runs on a benchtop mass spectrometer. The workflow is validated by a comparative analysis of BACE1-/- and wild-type mice using label-free quantification. The protease BACE1 cleaves the amyloid precursor protein (APP) as well as several other substrates and is a major drug target in Alzheimer's disease. We identified a total of 715 proteins with at least 2 unique peptides and quantified 522 of those proteins in CSF from BACE1-/- and wild-type mice. Several proteins, including the known BACE1 substrates APP, APLP1, CHL1 and contactin-2 showed lower abundance in the CSF of BACE1-/- mice, demonstrating that BACE1 substrate identification is possible from CSF. Additionally, ectonucleotide pyrophosphatase 5 was identified as a novel BACE1 substrate and validated in cells using immunoblots and by an in vitro BACE1 protease assay. Likewise, receptor-type tyrosine-protein phosphatase N2 and plexin domain-containing 2 were confirmed as BACE1 substrates by in vitro assays. Taken together, our study shows the deepest characterization of the mouse CSF proteome to date and the first quantitative analysis of the CSF proteome of individual mice. The BACE1 substrates identified in CSF may serve as biomarkers to monitor BACE1 activity in Alzheimer patients treated with BACE inhibitors.

  1. Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidence in rodent bioassays.

    Science.gov (United States)

    Paini, Alicia; Scholz, Gabriele; Marin-Kuan, Maricel; Schilter, Benoît; O'Brien, John; van Bladeren, Peter J; Rietjens, Ivonne M C M

    2011-09-01

    This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate whether a correlation can be obtained, using a benchmark dose (BMD) approach. Dose-response data on both carcinogenicity and in vivo DNA adduct formation were available for six compounds, i.e. 2-acetylaminofluorene, aflatoxin B1, methyleugenol, safrole, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and tamoxifen. BMD(10) values for liver carcinogenicity were calculated using the US Environmental Protection Agency BMD software. DNA adduct levels at this dose were extrapolated assuming linearity of the DNA adduct dose response. In addition, the levels of DNA adducts at the BMD(10) were compared to available data on endogenous background DNA damage in the target organ. Although for an individual carcinogen the tumour response increases when adduct levels increase, our results demonstrate that when comparing different carcinogens, no quantitative correlation exists between the level of DNA adduct formation and carcinogenicity. These data confirm that the quantity of DNA adducts formed by a DNA-reactive compound is not a carcinogenicity predictor but that other factors such as type of adduct and mutagenic potential may be equally relevant. Moreover, comparison to background DNA damage supports the notion that the mere occurrence of DNA adducts above or below the level of endogenous DNA damage is neither correlated to development of cancer. These data strongly emphasise the need to apply the mode of action framework to understand the contribution of other biological effect markers playing a role in carcinogenicity.

  2. In Vivo Quantitative Measurement of Arthritis Activity Based on Hydrophobically Modified Glycol Chitosan in Inflammatory Arthritis: More Active than Passive Accumulation

    Directory of Open Access Journals (Sweden)

    Kyeong Soon Park

    2012-09-01

    Full Text Available We demonstrated that arthritis could be visualized noninvasively using hydrophobically modified glycol chitosan nanoparticles labeled with Cy5.5 (HGC-Cy5.5 and an optical imaging system. Activated macrophages expressing Mac-1 molecules effectively phagocytosed HGC-Cy5.5, which formed spherical nanoparticles under physiologic conditions. We estimated the applicability of HGC-Cy5.5 to quantitative analysis of arthritis development and progression. Near-infrared fluorescence images, captured after HGC-Cy5.5 injection in mice with collagen-induced arthritis, showed stronger fluorescence intensity in the active arthritis group than in the nonarthritis group. According to the progression of arthritis in both collagen-induced arthritis and collagen antibody-induced arthritis models, total photon counts (TPCs increased in parallel with the clinical arthritis index. Quantitative analysis of fluorescence after treatment with methotrexate showed a significant decrease in TPC in a dose-dependent manner. Histologic evaluation confirmed that the mechanism underlying selective accumulation of HGC-Cy5.5 within synovitis tissues included enhanced phagocytosis of the probe by Mac-1-expressing macrophages as well as enhanced permeability through leaky vessels. These results suggest that optical imaging of arthritis using HGC-Cy5.5 can provide an objective measurement of disease activity and, at the same time, therapeutic responses in rheumatoid arthritis.

  3. Murine Gammaherpesvirus 68 ORF48 Is an RTA-Responsive Gene Product and Functions in both Viral Lytic Replication and Latency during In Vivo Infection.

    Science.gov (United States)

    Qi, Jing; Han, Chuanhui; Gong, Danyang; Liu, Ping; Zhou, Sheng; Deng, Hongyu

    2015-06-01

    Replication and transcription activator (RTA) of gammaherpesvirus is an immediate early gene product and regulates the expression of many downstream viral lytic genes. ORF48 is also conserved among gammaherpesviruses; however, its expression regulation and function remained largely unknown. In this study, we characterized the transcription unit of ORF48 from murine gammaherpesvirus 68 (MHV-68) and analyzed its transcriptional regulation. We showed that RTA activates the ORF48 promoter via an RTA-responsive element (48pRRE). RTA binds to 48pRRE directly in vitro and also associates with ORF48 promoter in vivo. Mutagenesis of 48pRRE in the context of the viral genome demonstrated that the expression of ORF48 is activated by RTA through 48pRRE during de novo infection. Through site-specific mutagenesis, we generated an ORF48-null virus and examined the function of ORF48 in vitro and in vivo. The ORF48-null mutation remarkably reduced the viral replication efficiency in cell culture. Moreover, through intranasal or intraperitoneal infection of laboratory mice, we showed that ORF48 is important for viral lytic replication in the lung and establishment of latency in the spleen, as well as viral reactivation from latency. Collectively, our study identified ORF48 as an RTA-responsive gene and showed that ORF48 is important for MHV-68 replication both in vitro and in vivo. The replication and transcription activator (RTA), conserved among gammaherpesviruses, serves as a molecular switch for the virus life cycle. It works as a transcriptional regulator to activate the expression of many viral lytic genes. However, only a limited number of such downstream genes have been uncovered for MHV-68. In this study, we identified ORF48 as an RTA-responsive gene of MHV-68 and mapped the cis element involved. By constructing a mutant virus that is deficient in ORF48 expression and through infection of laboratory mice, we showed that ORF48 plays important roles in different stages of

  4. Intranasal administration of human MSC for ischemic brain injury in the mouse: in vitro and in vivo neuroregenerative functions.

    Directory of Open Access Journals (Sweden)

    Vanessa Donega

    Full Text Available Intranasal treatment with C57BL/6 MSCs reduces lesion volume and improves motor and cognitive behavior in the neonatal hypoxic-ischemic (HI mouse model. In this study, we investigated the potential of human MSCs (hMSCs to treat HI brain injury in the neonatal mouse. Assessing the regenerative capacity of hMSCs is crucial for translation of our knowledge to the clinic. We determined the neuroregenerative potential of hMSCs in vitro and in vivo by intranasal administration 10 d post-HI in neonatal mice. HI was induced in P9 mouse pups. 1×10(6 or 2×10(6 hMSCs were administered intranasally 10 d post-HI. Motor behavior and lesion volume were measured 28 d post-HI. The in vitro capacity of hMSCs to induce differentiation of mouse neural stem cell (mNSC was determined using a transwell co-culture differentiation assay. To determine which chemotactic factors may play a role in mediating migration of MSCs to the lesion, we performed a PCR array on 84 chemotactic factors 10 days following sham-operation, and at 10 and 17 days post-HI. Our results show that 2×10(6 hMSCs decrease lesion volume, improve motor behavior, and reduce scar formation and microglia activity. Moreover, we demonstrate that the differentiation assay reflects the neuroregenerative potential of hMSCs in vivo, as hMSCs induce mNSCs to differentiate into neurons in vitro. We also provide evidence that the chemotactic factor CXCL10 may play an important role in hMSC migration to the lesion site. This is suggested by our finding that CXCL10 is significantly upregulated at 10 days following HI, but not at 17 days after HI, a time when MSCs no longer reach the lesion when given intranasally. The results described in this work also tempt us to contemplate hMSCs not only as a potential treatment option for neonatal encephalopathy, but also for a plethora of degenerative and traumatic injuries of the nervous system.

  5. Folic acid-functionalized up-conversion nanoparticles: toxicity studies in vivo and in vitro and targeted imaging applications

    Science.gov (United States)

    Sun, Lining; Wei, Zuwu; Chen, Haige; Liu, Jinliang; Guo, Jianjian; Cao, Ming; Wen, Tieqiao; Shi, Liyi

    2014-07-01

    Folate receptors (FRs) are overexpressed on a variety of human cancer cells and tissues, including cancers of the breast, ovaries, endometrium, and brain. This over-expression of FRs can be used to target folate-linked imaging specifically to FR-expressing tumors. Fluorescence is emerging as a powerful new modality for molecular imaging in both the diagnosis and treatment of disease. Combining innovative molecular biology and chemistry, we prepared three kinds of folate-targeted up-conversion nanoparticles as imaging agents (UCNC-FA: UCNC-Er-FA, UCNC-Tm-FA, and UCNC-Er,Tm-FA). In vivo and in vitro toxicity studies showed that these nanoparticles have both good biocompatibility and low toxicity. Moreover, the up-conversion luminescence imaging indicated that they have good targeting to HeLa cells and can therefore serve as potential fluorescent contrast agents.Folate receptors (FRs) are overexpressed on a variety of human cancer cells and tissues, including cancers of the breast, ovaries, endometrium, and brain. This over-expression of FRs can be used to target folate-linked imaging specifically to FR-expressing tumors. Fluorescence is emerging as a powerful new modality for molecular imaging in both the diagnosis and treatment of disease. Combining innovative molecular biology and chemistry, we prepared three kinds of folate-targeted up-conversion nanoparticles as imaging agents (UCNC-FA: UCNC-Er-FA, UCNC-Tm-FA, and UCNC-Er,Tm-FA). In vivo and in vitro toxicity studies showed that these nanoparticles have both good biocompatibility and low toxicity. Moreover, the up-conversion luminescence imaging indicated that they have good targeting to HeLa cells and can therefore serve as potential fluorescent contrast agents. Electronic supplementary information (ESI) available: Up-conversion luminescence spectra of UCNC-Er and UCNC-Er-FA, UCNC-Tm and UCNC-Tm-FA. Confocal luminescence imaging data collected as a series along the Z optical axis. See DOI: 10.1039/c4nr02312a

  6. In vivo measurement of the shape of the tissue-refractive-index correlation function and its applicationto detection of colorectal field carcinogenesis

    Science.gov (United States)

    Gomes, Andrew J.; Ruderman, Sarah; DelaCruz, Mart; Wali, Ramesh K.; Roy, Hemant K.; Backman, Vadim

    2012-04-01

    Polarization-gated spectroscopy is an established method to depth-selectively interrogate the structural properties of biological tissue. We employ this method in vivo in the azoxymethane (AOM)-treated rat model to monitor the morphological changes that occur in the field of a tumor during early carcinogenesis. The results demonstrate a statistically significant change in the shape of the refractive-index correlation function for AOM-treated rats versus saline-treated controls. Since refractive index is linearly proportional to mass density, these refractive-index changes can be directly linked to alterations in the spatial distribution patterns of macromolecular density. Furthermore, we found that alterations in the shape of the refractive-index correlation function shape were an indicator of both present and future risk of tumor development. These results suggest that noninvasive measurement of the shape of the refractive-index correlation function could be a promising marker of early cancer development.

  7. Gene transfer in rodents and primates as a new tool for modeling diseases in animals and assessing functions by in vivo imaging

    Energy Technology Data Exchange (ETDEWEB)

    Deglon, N. [Atomic Energy Commission (CEA), Dept. of Medical Research and MIRCen Program, 91 - Orsay (France)

    2006-07-01

    The identification of disease-causing genes in familial forms of neuro-degenerative disorders and the development of genetic models closely replicating human CNS pathologies have drastically changed our understanding of the molecular events leading to neuronal cell death. If these achievements open new opportunities of therapeutic interventions efficient delivery systems taking into account the specificity of the central nervous system are required to administer therapeutic candidates. In addition, there is a need to develop 1) genetic models in large animals that replicate late stages of the diseases and 2) imaging techniques suitable for longitudinal, quantitative and non-invasive evaluation of disease progression and the evaluation of new therapeutic strategies. Over the last few years, we have investigated the potential of lentiviral vectors as tool to model and treat CNS disorders. The use of lentiviral vectors to create animal model of these pathologies holds various advantages compared to classical transgenic approaches. Viral vectors are versatile, highly flexible tools to perform in vivo studies. Multiple genetic models can be created in a short period of time. High transduction efficiencies as well as robust and sustained trans-gene expression lead to the rapid appearance of functional and behavioral abnormalities and severe neuro-degeneration. Targeted injections in different brain areas can be used to investigate the regional specificity of the neuro-pathology and eliminate potential side effects associated with a widespread over-expression of the trans-gene. Finally, models can be established in different mammalian species including non-human primates, thereby providing an opportunity to assess complex behavioral changes and perform longitudinal follow-up of neuro-pathological alterations by imaging. We have demonstrated the proof of principle of this approach for Huntington's disease. We have shown that the intratriatal injection of lentiviral

  8. Quantitative proteomic analysis reveals that anti-cancer effects of selenium-binding protein 1 in vivo are associated with metabolic pathways.

    Science.gov (United States)

    Ying, Qi; Ansong, Emmanuel; Diamond, Alan M; Lu, Zhaoxin; Yang, Wancai; Bie, Xiaomei

    2015-01-01

    Previous studies have shown the tumor-suppressive role of selenium-binding protein 1 (SBP1), but the underlying mechanisms are unclear. In this study, we found that induction of SBP1 showed significant inhibition of colorectal cancer cell growth and metastasis in mice. We further employed isobaric tags for relative and absolute quantitation (iTRAQ) to identify proteins that were involved in SBP1-mediated anti-cancer effects in tumor tissues. We identified 132 differentially expressed proteins, among them, 53 proteins were upregulated and 79 proteins were downregulated. Importantly, many of the differentially altered proteins were associated with lipid/glucose metabolism, which were also linked to Glycolysis, MAPK, Wnt, NF-kB, NOTCH and epithelial-mesenchymal transition (EMT) signaling pathways. These results have revealed a novel mechanism that SBP1-mediated cancer inhibition is through altering lipid/glucose metabolic signaling pathways.

  9. Quantitative autoradiography of ligands for dopamine receptors and transporters in brain of Göttingen minipig: comparison with results in vivo

    DEFF Research Database (Denmark)

    Minuzzi, Luciano; Alstrup, Aage Kristian Olsen; Bender, Dirk

    2006-01-01

    The pig has been used as animal model for positron emission tomography (PET) studies of dopamine (DA) receptors and pharmacological perturbations of DA neurotransmission. However, the binding properties of DA receptors and transporters in pig brain have not been characterized in vitro. Therefore......, the saturation binding parameters of [3H]SCH 23390 for DA D1 receptors and [3H]raclopride for DA D2/3 receptors were measured by quantitative autoradiography in cryostat sections from brain of groups of 8 week old and adult female Göttingen minipigs. The magnitudes of Bmax and Kd for these ligands were similar...... in young and old pigs, and were close to those reported for rat and human brain. Furthermore, gradients in the concentrations of D1 and D2/3 sites in striatum measured in vitro agreed with earlier findings in PET studies. However, the dopamine transporter (DAT) ligand [3H]GBR12935 did not bind in pig brain...

  10. In vivo redundant function of the 3' IgH regulatory element HS3b in the mouse.

    Science.gov (United States)

    Bébin, Anne-Gaëlle; Carrion, Claire; Marquet, Marie; Cogné, Nadine; Lecardeur, Sandrine; Cogné, Michel; Pinaud, Eric

    2010-04-01

    In the mouse, the regulatory region located at the 3' end of the IgH locus includes four transcriptional enhancers: HS3a, HS1-2, HS3b, and HS4; the first three lie in a quasi-palindromic structure. Although the upstream elements HS3a and HS1-2 proved dispensable for Ig expression and class switch recombination (CSR), the joint deletion of HS3b and HS4 led to a consistent decrease in IgH expression in resting B cells and to a major CSR defect. Within this pair of distal enhancers, it was questionable whether HS3b and HS4 could be considered individually as elements critical for IgH expression and/or CSR. Studies in HS4-deficient mice recently revealed the role of HS4 as restricted to Igmicro-chain expression from the pre-B to the mature B cell stage and left HS3b as the last candidate for CSR regulation. Our present study finally invalidates the hypothesis that CSR could mostly rely on HS3b itself. B cells from HS3b-deficient animals undergo normal proliferation, germline transcription, and CSR upon in vitro stimulation with LPS; in vivo Ag-specific responses are not affected. In conclusion, our study highlights a major effect of the global ambiance of the IgH locus; enhancers demonstrated as being strongly synergistic in transgenes turn out to be redundant in their endogenous context.

  11. In-vivo cutaneous burn characterization and scar assay with multi-functional optical coherence tomography (Conference Presentation)

    Science.gov (United States)

    Kim, Bumju; Yoon, Yeorum; Le, Viet-Hoan; Yoon, Calvin J.; Kim, Ki Hean

    2016-03-01

    Research about the cutaneous burn was separated by assessment of burn depth and development of wound healing therapy. Various in vivo optical techniques were used to determined burn depth and observe the wound healing process. In this paper, we report the usage of multimodal optical coherence tomography system, which containing angiographic and polarization sensitive OCT (PS-OCT) with conventional OCT system, at burn studies. Burn was induced at 4 different degrees by control the attachment time of 75 Celsius degree heated brass rod at dorsal skin of the rat. For the burn depth assessment, we imaged the different burn degrees area. Changes of polarization sensitive signal were providing burn depth information. To see the wound healing process, each wound area imaged at long period. Conventional OCT shows the structural information about the tissue, like layer and hair follicle. Angiographic OCT provides vascular distribution and diameter of blood vessel information and PS-OCT shows birefringence tissue information. Based on the multimodal OCT data, burn depth assessment were well matched with burn induced time and wound healing process was consistent with previous wound healing report. Therefore, the multimodal OCT holds potential for burn study.

  12. Creating frog heart as an organ: in vitro-induced heart functions as a circulatory organ in vivo.

    Science.gov (United States)

    Kinoshita, Masayoshi; Ariizumi, Takashi; Yuasa, Shinsuke; Miyoshi, Shunichirou; Komazaki, Shinji; Fukuda, Keiichi; Asashima, Makoto

    2010-01-01

    Cardiomyocytes have been induced from various pluripotent cells, such as embryonic stem cells and myeloid stem cells; however, the generation of cardiac tissues beyond two-dimensional cell-sheets has not been reported. Creating higher order, three-dimensional structures that are unique to heart is the long-awaited next step in realizing cardiac regenerative medicine. We have previously shown that cardiomyocytes can be induced in vitro from undifferentiated cells (animal caps) excised from Xenopus embryos. Cardiomyocytes were induced by first dissociating the animal caps and then reaggregating them following treatment with activin. Here, we describe an interesting method for creating a complete ectopic heart in vivo, involving the introduction of in vitro-created tissue during early embryogenesis. Thus, animal cap reaggregates were transplanted into the abdomen of late-neurula-stage embryos, resulting in two-chambered hearts being formed. The dual-heart larvae matured into adult animals with transplanted hearts intact. Involvement of transplanted hearts in systemic circulation was demonstrated. Moreover, the ectopic hearts possessed higher order structures such as atrium and ventricle, and were morphologically, histologically, and electrophysiologically identical to original hearts. This system should facilitate the study of heart organogenesis and may promote a shift from tissue to organ engineering for clinical applications.

  13. Quantitative Reasoning and the Sine Function: The Case of Zac

    Science.gov (United States)

    Moore, Kevin C.

    2014-01-01

    A growing body of literature has identified quantitative and covariational reasoning as critical for secondary and undergraduate student learning, particularly for topics that require students to make sense of relationships between quantities. The present study extends this body of literature by characterizing an undergraduate precalculus…

  14. Quantitative Reasoning and the Sine Function: The Case of Zac

    Science.gov (United States)

    Moore, Kevin C.

    2014-01-01

    A growing body of literature has identified quantitative and covariational reasoning as critical for secondary and undergraduate student learning, particularly for topics that require students to make sense of relationships between quantities. The present study extends this body of literature by characterizing an undergraduate precalculus…

  15. The human lactase persistence-associated SNP -13910*T enables in vivo functional persistence of lactase promoter-reporter transgene expression.

    Science.gov (United States)

    Fang, Lin; Ahn, Jong Kun; Wodziak, Dariusz; Sibley, Eric

    2012-07-01

    Lactase is the intestinal enzyme responsible for digestion of the milk sugar lactose. Lactase gene expression declines dramatically upon weaning in mammals and during early childhood in humans (lactase nonpersistence). In various ethnic groups, however, lactase persists in high levels throughout adulthood (lactase persistence). Genetic association studies have identified that lactase persistence in northern Europeans is strongly associated with a single nucleotide polymorphism (SNP) located 14 kb upstream of the lactase gene: -13910*C/T. To determine whether the -13910*T SNP can function in vivo to mediate lactase persistence, we generated transgenic mice harboring human DNA fragments with the -13910*T SNP or the ancestral -13910*C SNP cloned upstream of a 2-kb rat lactase gene promoter in a luciferase reporter construct. We previously reported that the 2-kb rat lactase promoter directs a post-weaning decline of luciferase transgene expression similar to that of the endogenous lactase gene. In the present study, the post-weaning decline directed by the rat lactase promoter is impeded by addition of the -13910*T SNP human DNA fragment, but not by addition of the -13910*C ancestral SNP fragment. Persistence of transgene expression associated with the -13910*T SNP represents the first in vivo data in support of a functional role for the -13910*T SNP in mediating the human lactase persistence phenotype.

  16. Longitudinal, 3D in vivo imaging of sebaceous glands by coherent anti-Stokes Raman scattering microscopy –normal function and response to cryotherapy

    Science.gov (United States)

    Jung, Yookyung; Tam, Joshua; Jalian, H. Ray; Anderson, R. Rox; Evans, Conor L.

    2014-01-01

    Sebaceous glands perform complex functions, and are centrally involved in the pathogenesis of acne vulgaris. Current techniques for studying sebaceous glands are mostly static in nature, whereas the gland’s main function – excretion of sebum via the holocrine mechanism – can only be evaluated over time. We present a longitudinal, real-time alternative – the in vivo, label-free imaging of sebaceous glands using Coherent Anti-Stokes Raman Scattering (CARS) microscopy, which is used to selectively visualize lipids. In mouse ears, CARS microscopy revealed dynamic changes in sebaceous glands during the holocrine secretion process, as well as in response to damage to the glands caused by cooling. Detailed gland structure, plus the active migration of individual sebocytes and cohorts of sebocytes were measured. Cooling produced characteristic changes in sebocyte structure and migration. This study demonstrates that CARS microscopy is a promising tool for studying the sebaceous gland and its associated disorders in three-dimensions in vivo. PMID:25026458

  17. Chronic In Vivo Interaction of Dendritic Cells Expressing the Ligand Rae-1ε with NK Cells Impacts NKG2D Expression and Function.

    Science.gov (United States)

    Morvan, Maelig G; Champsaur, Marine; Reizis, Boris; Lanier, Lewis L

    2017-05-01

    To investigate how dendritic cells (DCs) interact with NK cells in vivo, we developed a novel mouse model in which Rae-1ε, a ligand of the NKG2D receptor, is expressed in cells with high levels of CD11c. In these CD11c-Rae1 mice, expression of Rae-1 was confirmed on all subsets of DCs and a small subset of B and T cells, but not on NK cells. DC numbers and activation status were unchanged, and NK cells in these CD11c-Rae1 mice presented the same Ly49 repertoire and maturation levels as their littermate wildtype controls. Early NK cell activation after mouse CMV infection was slightly lower than in wildtype mice, but NK cell expansion and viral control were comparable. Notably, we demonstrate that chronic interaction of NK cells with NKG2D ligand-expressing DCs leads to a reversible NKG2D down-modulation, as well as impaired NKG2D-dependent NK cell functions, including tumor rejection. In addition to generating a useful mouse model, our studies reveal in vivo the functional importance of the NK cell and DC cross-talk.

  18. Functional membrane androgen receptors in colon tumors trigger pro-apoptotic responses in vitro and reduce drastically tumor incidence in vivo

    Directory of Open Access Journals (Sweden)

    Föller Michael

    2009-12-01

    Full Text Available Abstract Background Membrane androgen receptors (mAR have been implicated in the regulation of cell growth, motility and apoptosis in prostate and breast cancer. Here we analyzed mAR expression and function in colon cancer. Results Using fluorescent mAR ligands we showed specific membrane staining in colon cell lines and mouse xenograft tumor tissues, while membrane staining was undetectable in healthy mouse colon tissues and non-transformed intestinal cells. Saturation/displacement assays revealed time- and concentration-dependent specific binding for testosterone with a KD of 2.9 nM. Stimulation of colon mAR by testosterone albumin conjugates induced rapid cytoskeleton reorganization and apoptotic responses, even in the presence of anti-androgens. The actin cytoskeleton drug cytochalasin B effectively inhibited the pro-apoptotic responses and caspase-3 activation. Interestingly, in vivo studies revealed that mAR activation resulted in a 65% reduction of tumor incidence in chemically induced Balb/c mice colon tumors. Conclusion Our results demonstrate for the first time that functional mARs are predominantly expressed in colon tumors and that their activation results in induction of anti-tumor responses in vitro and extensive reduction of tumor incidence in vivo.

  19. Amnionless function is required for cubilin brush-border expression and intrinsic factor-cobalamin (vitamin B12) absorption in vivo.

    Science.gov (United States)

    He, Qianchuan; Madsen, Mette; Kilkenney, Adam; Gregory, Brittany; Christensen, Erik I; Vorum, Henrik; Højrup, Peter; Schäffer, Alejandro A; Kirkness, Ewen F; Tanner, Stephan M; de la Chapelle, Albert; Giger, Urs; Moestrup, Søren K; Fyfe, John C

    2005-08-15

    Amnionless (AMN) and cubilin gene products appear to be essential functional subunits of an endocytic receptor called cubam. Mutation of either gene causes autosomal recessive Imerslund-Gräsbeck syndrome (I-GS, OMIM no. 261100) in humans, a disorder characterized by selective intestinal malabsorption of cobalamin (vitamin B12) and urinary loss of several specific low-molecular-weight proteins. Vital insight into the molecular pathology of I-GS has been obtained from studies of dogs with a similar syndrome. In this work, we show that I-GS segregates in a large canine kindred due to an in-frame deletion of 33 nucleotides in exon 10 of AMN. In a second, unrelated I-GS kindred, affected dogs exhibit a homozygous substitution in the AMN translation initiation codon. Studies in vivo demonstrated that both mutations abrogate AMN expression and block cubilin processing and targeting to the apical membrane. The essential features of AMN dysfunction observed in vivo are recapitulated in a heterologous cell-transfection system, thus validating the system for analysis of AMN-cubilin interactions. Characterization of canine AMN mutations that cause I-GS establishes the canine model as an ortholog of the human disorder well suited to studies of AMN function and coevolution with cubilin.

  20. Noninvasive targeting delivery and in vivo magnetic resonance tracking method for live apoptotic cells in cerebral ischemia with functional Fe2O3 magnetic nanoparticles.

    Science.gov (United States)

    Saito, Atsushi; Mekawy, Moataz M; Sumiyoshi, Akira; Riera, Jorge J; Shimizu, Hiroaki; Kawashima, Ryuta; Tominaga, Teiji

    2016-03-11

    Apoptotic neuronal death is known as programmed cell death. Inhibition of this progression might contribute to a new treatment strategy. However, methods for in vivo detection of live apoptotic cells are in need to be developed and established. The purpose of this study is to develop a new method for in vivo brain imaging for live apoptotic lesions using magnetic resonance imaging (MRI). We focused on the specific accumulation of our recently developed functional magnetic nanoparticles (FMNPs) into apoptotic cells using a rat cerebral ischemia model. Sulphorhodamine B, fluorescent dye was linked to valylalanylaspartic acid fluoromethyl ketone as a pan-caspase inhibitor to form SR-FLIVO. SR-FLIVO was bound with FMNPs to develop SR-FLIVO-FMNP probe. Ischemic rat brains were scanned by 7T MRI before and after intravenous injection of SR-FLIVO-FMNP and the distribution was evaluated by subtraction images of T2* colored mapping. SR-FLIVO, intracellular FMNPs, and T2* reduction area were histologically analyzed. The distribution of SR-FLIVO-FMNP was evaluated by subtracting the T2* signal images and was significantly correlated with the histological findings by TUNEL staining. Our experimental results revealed several findings where our newly developed probe SR-FLIVO-FMNP was intravenously administered into ischemic rats and FLIVO expression was tracked and found in apoptotic cells in rat brains after cerebral ischemia. A remarkable T2* reduction within the ischemic lesion was recorded using MRI based SR-FLIVO-FMNP probe as a contrasting agent due to the specific probe accumulation in apoptotic cells whereas, no observation of T2* reduction within the non-ischemic lesion due to no probe accumulation in non-apoptotic cells. Histological analysis based on the correlation between FLIVO and TUNEL staining showed that almost all FLIVO-positive cells were positive for TUNEL staining. These findings suggest the possibility for establishment of in vivo targeting delivery

  1. In vivo mitochondrial function in HIV-infected persons treated with contemporary anti-retroviral therapy: a magnetic resonance spectroscopy study.

    Directory of Open Access Journals (Sweden)

    Brendan A I Payne

    Full Text Available Modern anti-retroviral therapy is highly effective at suppressing viral replication and restoring immune function in HIV-infected persons. However, such individuals show reduced physiological performance and increased frailty compared with age-matched uninfected persons. Contemporary anti-retroviral therapy is thought to be largely free from neuromuscular complications, whereas several anti-retroviral drugs previously in common usage have been associated with mitochondrial toxicity. It has recently been established that patients with prior exposure to such drugs exhibit irreversible cellular and molecular mitochondrial defects. However the functional significance of such damage remains unknown. Here we use phosphorus magnetic resonance spectroscopy ((31P-MRS to measure in vivo muscle mitochondrial oxidative function, in patients treated with contemporary anti-retroviral therapy, and compare with biopsy findings (cytochrome c oxidase (COX histochemistry. We show that dynamic oxidative function (post-exertional ATP (adenosine triphosphate resynthesis was largely maintained in the face of mild to moderate COX defects (affecting up to ∼10% of fibers: τ½ ADP (half-life of adenosine diphosphate clearance, HIV-infected 22.1±9.9 s, HIV-uninfected 18.8±4.4 s, p = 0.09. In contrast, HIV-infected patients had a significant derangement of resting state ATP metabolism compared with controls: ADP/ATP ratio, HIV-infected 1.24±0.08×10(-3, HIV-uninfected 1.16±0.05×10(-3, p = 0.001. These observations are broadly reassuring in that they suggest that in vivo mitochondrial function in patients on contemporary anti-retroviral therapy is largely maintained at the whole organ level, despite histochemical (COX defects within individual cells. Basal energy requirements may nevertheless be increased.

  2. Hexarelin Protects Rodent Pancreatic Β-Cells Function from Cytotoxic Effects of Streptozotocin Involving Mitochondrial Signalling Pathways In Vivo and In Vitro.

    Directory of Open Access Journals (Sweden)

    Yan Zhao

    Full Text Available Mitochondrial functions are crucial for pancreatic β-cell survival and glucose-induced insulin secretion. Hexarelin (Hex is a synthetic small peptide ghrelin analogue, which has been shown to protect cardiomyocytes from the ischemia-reperfusion process. In this study, we used in vitro and in vivo models of streptozotocin (STZ-induced β-cell damage to study the protective effect of Hex and the associated mechanisms. We found that STZ produced a cytotoxic effect in a dose- and time-dependent manner in MIN6 cells (a mouse β-cell line. Hex (1.0 μM decreased the STZ-induced damage in β-cells. Rhodamine 123 assay and superoxide DHE production assay revealed that Hex ameliorated STZ-induced mitochondrial damage and excessive superoxide activity in β-cells. In addition, Hex significantly reduced STZ-induced expression of cleaved Caspases-3, Caspases-9 and the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2 in MIN6 cells. We further examined the in vivo effect of Hex in a rat model of type 1 diabetes induced by STZ injection. Hex ameliorated STZ-induced decrease in plasma insulin and protected the structure of islets from STZ-induced disruption. Hex also ameliorated STZ-induced expression of cleaved Caspase-9 and the Bax in β-cells. In conclusion, our data indicate that Hex is able to protects β-cell mass from STZ-caused cytotoxic effects involving mitochondrial pathways in vitro and in vivo. Hex may serve as a potential protective agent for the management of diabetes.

  3. Regional brain metabolite abnormalities in inherited prion disease and asymptomatic gene carriers demonstrated in vivo by quantitative proton magnetic resonance spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Waldman, A.D.; Cordery, R.J.; Godbolt, A.; Rossor, M.N. [University College London, Dementia Research Group, Department of Neurodegenerative Disease, Institute of Neurology, London (United Kingdom); Imperial College of Science, Technology and Medicine, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, London (United Kingdom); MacManus, D.G. [University College London, NMR Research Unit, Department of Clinical Neurology, Institute of Neurology, London (United Kingdom); Collinge, J. [University College London, MRC Prion Unit, Department of Neurodegenerative Disease, Institute of Neurology, London (United Kingdom)

    2006-06-15

    Inherited prion diseases are caused by mutations in the gene which codes for prion protein (PrP), leading to proliferation of abnormal PrP isomers in the brain and neurodegeneration; they include Gerstmann-Straeussler-Scheinker disease (GSS), fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). We studied two patients with symptomatic inherited prion disease (P102L) and two pre-symptomatic P102L gene carriers using quantitative magnetic resonance spectroscopy (MRS). Short echo time spectra were acquired from the thalamus, caudate region and frontal white matter, metabolite levels and ratios were measured and z-scores calculated for individual patients relative to age-matched normal controls. MRS data were compared with structural magnetic resonance imaging. One fCJD case had generalised atrophy and showed increased levels of myo-inositol (MI) in the thalamus (z=3.7). The other had decreased levels of N-acetylaspartate (z=4) and diffuse signal abnormality in the frontal white matter. Both asymptomatic gene carriers had normal imaging, but increased frontal white matter MI (z=4.3, 4.1), and one also had increased MI in the caudate (z=5.3). Isolated MI abnormalities in asymptomatic gene carriers are a novel finding and may reflect early glial proliferation, prior to significant neuronal damage. MRS provides potential non-invasive surrogate markers of early disease and progression in inherited prion disease. (orig.)

  4. Development, evaluation, and in-vivo validation of two non-invasive methods for quantitation of activity and dosimetry of monoclonal antibodies in humans

    Energy Technology Data Exchange (ETDEWEB)

    Hammond, N.D.; Moldofsky, P.J.; Exten, R.E.; Gatenby, R.A.; Broder, G.J.

    1985-05-01

    The authors have applied both a conjugate view imaging method and a first pass study for quantitation of absolute I-131 activity in lesions and normal tissue of patients with colon carcinoma in order to study biological clearance of the I-131 F(ab)'/sub 2/ fragments of mouse monoclonal antibody and the resultant dosimetry. Both methods require a transmission scan for determining patient attenuation and measurement of patient lesion or organ size in the region of interest. The conjugate view method is analyzed for both SPECT and planar imaging. The percent error of both methods relates to lesion size and absolute activity when compared to actual well-counter assayed samples of malignant and normal tissue obtained from CT-guided needle biopsies or surgical specimens. Dosimetric evaluation was based on determination of activity, clearance from computer-generated time-activity curves and lesion or organ volumes from volumetric CT scan data. The dose to the thyroid gland was calculated for one population receiving Lugol's solution 3 days prior and for the other who received Lugol's at the time of administration. Data showed no significant difference in absorbed thyroid dose. Lastly, the absolute uptake of I-131, lesion to background ratios, and the dosimetry data were compared for three different monoclonal antibody fragments.

  5. Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

    Science.gov (United States)

    Nebl, Thomas; Prieto, Judith Helena; Kapp, Eugene; Smith, Brian J.; Williams, Melanie J.; Yates, John R.; Cowman, Alan F.; Tonkin, Christopher J.

    2011-01-01

    Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca2+-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of 32[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca2+-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component. PMID:21980283

  6. Overcoming the heterologous bias: An in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata

    Energy Technology Data Exchange (ETDEWEB)

    Puri, Nidhi; Manoharlal, Raman; Sharma, Monika [Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067 (India); Sanglard, Dominique [Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne (Switzerland); Prasad, Rajendra, E-mail: rp47jnu@gmail.com [Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067 (India)

    2011-01-07

    Research highlights: {yields} First report to demonstrate an in vivo expression system of an ABC multidrug transporter CgCdr1p of C. glabrata. {yields} First report on the structure and functional characterization of CgCdr1p. {yields} Functional conservation of divergent but typical residues of CgCdr1p. {yields} CgCdr1p elicits promiscuity towards substrates and has a large drug binding pocket with overlapping specificities. -- Abstract: We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the arte-factual concerns

  7. Antibodies to Staphylococcus aureus capsular polysaccharides 5 and 8 perform similarly in vitro but are functionally distinct in vivo.

    Science.gov (United States)

    Liu, Bo; Park, Saeyoung; Thompson, Christopher D; Li, Xue; Lee, Jean C

    2016-12-09

    The capsular polysaccharide (CP) produced by Staphylococcus aureus is a virulence factor that allows the organism to evade uptake and killing by host neutrophils. Polyclonal antibodies to the serotype 5 (CP5) and type 8 (CP8) capsular polysaccharides are opsonic and protect mice against experimental bacteremia provoked by encapsulated staphylococci. Thus, passive immunotherapy using CP antibodies has been considered for the prevention or treatment of invasive antibiotic-resistant S. aureus infections. In this report, we generated monoclonal antibodies (mAbs) against S. aureus CP5 or CP8. Backbone specific mAbs reacted with native and O-deacetylated CPs, whereas O-acetyl specific mAbs reacted only with native CPs. Reference strains of S. aureus and a selection of clinical isolates reacted by colony immunoblot with the CP5 and CP8 mAbs in a serotype-specific manner. The mAbs mediated in vitro CP type-specific opsonophagocytic killing of S. aureus strains, and mice passively immunized with CP5 mAbs were protected against S. aureus bacteremia. Neither CP8-specific mAbs or polyclonal antibodies protected mice against bacteremia provoked by serotype 8 S. aureus clinical isolates, although these same antibodies did protect against a serotype 5 S. aureus strain genetically engineered to produce CP8. We detected soluble CP8 in culture supernatants of serotype 8 clinical isolates and in the plasma of infected animals. Serotype 5 S. aureus released significantly less soluble CP5 in vitro and in vivo. The release of soluble CP8 by S. aureus may contribute to the inability of CP8 vaccines or antibodies to protect against serotype 8 staphylococcal infections.

  8. Regulation of in vitro and in vivo immune functions by the cytosolic adaptor protein SKAP-HOM.

    Science.gov (United States)

    Togni, M; Swanson, K D; Reimann, S; Kliche, S; Pearce, A C; Simeoni, L; Reinhold, D; Wienands, J; Neel, B G; Schraven, B; Gerber, A

    2005-09-01

    SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOM-deficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca(2+) responses, are normal in SKAP-HOM(-/-) animals. However, despite apparently normal membrane-proximal signaling events, BCR-mediated proliferation is strongly attenuated in the absence of SKAP-HOM(-/-). In addition, adhesion of activated B cells to fibronectin (a ligand for beta1 integrins) as well as to ICAM-1 (a ligand for beta2 integrins) is strongly reduced. In vivo, the loss of SKAP-HOM results in a less severe clinical course of experimental autoimmune encephalomyelitis following immunization of mice with the encephalitogenic peptide of MOG (myelin oligodendrocyte glycoprotein). This is accompanied by strongly reduced serum levels of MOG-specific antibodies and lower MOG-specific T-cell responses. In summary, our data suggest that SKAP-HOM is required for proper activation of the immune system, likely by regulating the cross-talk between immunoreceptors and integrins.

  9. Quantitative assessment of regional right ventricular function with color kinesis.

    Science.gov (United States)

    Vignon, P; Weinert, L; Mor-Avi, V; Spencer, K T; Bednarz, J; Lang, R M

    1999-06-01

    We used color kinesis, a recent echocardiographic technique that provides regional information on the magnitude and timing of endocardial wall motion, to quantitatively assess regional right ventricular (RV) systolic and diastolic properties in 76 subjects who were divided into five groups, as follows: normal (n = 20), heart failure (n = 15), pressure/volume overload (n = 14), pressure overload (n = 12), and RV hypertrophy (n = 15). Quantitative segmental analysis of color kinesis images was used to obtain regional fractional area change (RFAC), which was displayed in the form of stacked histograms to determine patterns of endocardial wall motion. Time curves of integrated RFAC were used to objectively identify asynchrony of diastolic endocardial motion. When compared with normal subjects, patients with pressure overload or heart failure exhibited significantly decreased endocardial motion along the RV free wall. In the presence of mixed pressure/volume overload, the markedly increased ventricular septal motion compensated for decreased RV free wall motion. Diastolic endocardial wall motion was delayed in 17 of 72 segments (24%) in patients with RV pressure overload, and in 31 of 90 segments (34%) in patients with RV hypertrophy. Asynchrony of diastolic endocardial wall motion was greater in the latter group than in normal subjects (16% versus 10%: p kinesis images allows quantitative assessment of regional RV systolic and diastolic properties.

  10. Carbon nanotubes for in vivo cancer nanotechnology

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The latest progress of using carbon nanotubes(CNTs) for in vivo cancer nanotechnology is reviewed.CNTs can be functionalized by either covalent or non-covalent chemistry to produce functional bioconjugates for many in vivo applications.In vivo behaviors and toxicology studies of CNTs are summarized,suggesting no significant toxicity of well functionalized CNTs to the treated mice.Owing to their unique chemical and physical properties,CNTs,especially single-walled carbon nanotubes(SWNTs),have been widely used for various modalities of in vivo cancer treatment and imaging.Future development of CNT-based nanomedicine may bring novel opportunities to cancer diagnosis and therapy.

  11. P3 optimization of functional potency, in vivo efficacy and oral bioavailability in 3-aminopyrazinone thrombin inhibitors bearing non-charged groups at the P1 position.

    Science.gov (United States)

    Isaacs, Richard C A; Newton, Christina L; Cutrona, Kellie J; Mercer, Swati P; Dorsey, Bruce D; McDonough, Colleen M; Cook, Jacquelynn J; Krueger, Julie A; Lewis, S Dale; Lucas, Bobby J; Lyle, Elizabeth A; Lynch, Joseph J; Miller-Stein, Cynthia; Michener, Maria T; Wallace, Audrey A; White, Rebecca B; Wong, Bradley K

    2011-03-01

    Although the S3 pocket of the thrombin active site is lined with lipophilic amino acid residues, the accommodation of polarity within the lipophilic P3 moiety of small molecule inhibitors is possible provided that the polar functionality is capable of pointing away from the binding pocket outwards toward solvent while simultaneously allowing the lipophilic portion of the P3 ligand to interact with the S3 amino acid residues. Manipulation of this motif provided the means to effect optimization of functional potency, in vivo antithrombotic efficacy and oral bioavailability in a series of 3-aminopyrazinone thrombin inhibitors which contained non-charged groups at the P1 position. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. In vivo secretory potential and the effect of combination therapy with octreotide and cabergoline in patients with clinically non-functioning pituitary adenomas

    DEFF Research Database (Denmark)

    Andersen, M; Bjerre, P; Schrøder, H D;

    2001-01-01

    The secretory capacity, in vivo, of clinically non-functioning pituitary adenomas may possibly predict tumour volume reduction during intensive medical therapy. Ten patients (mean (range) 53 years (26-73)) with clinically non-functioning macroadenomas, > or = 10 mm were studied. The secretory...... capacity of the adenomas was examined using basal, NaCl and TRH-stimulated LH, FSH and alpha-subunit levels. The effect on tumour volume of 6 months' therapy with the combination of a somatostatin analogue, octreotide 200 microg x 3/day and a dopamine-D2-agonist, cabergoline 0.5 mg x 1/day was studied....... The basal LH, FSH and alpha-subunit levels were determined before and during 6 months' therapy with octreotide and cabergoline, and MR scans were used to evaluate tumour volume before and during this period of therapy. Octopus-perimetry was used to examine the visual fields. A reduction in tumour volume...

  13. Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex.

    Directory of Open Access Journals (Sweden)

    Thomas Nebl

    2011-09-01

    Full Text Available Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca²⁺-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of ³²[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca²⁺-dependent phosphorylation patterns on three of its components--GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.

  14. Hyperspectral Imaging Using Intracellular Spies: Quantitative Real-Time Measurement of Intracellular Parameters In Vivo during Interaction of the Pathogenic Fungus Aspergillus fumigatus with Human Monocytes

    Science.gov (United States)

    Mohebbi, Sara; Erfurth, Florian; Hennersdorf, Philipp; Brakhage, Axel A.; Saluz, Hans Peter

    2016-01-01

    Hyperspectral imaging (HSI) is a technique based on the combination of classical spectroscopy and conventional digital image processing. It is also well suited for the biological assays and quantitative real-time analysis since it provides spectral and spatial data of samples. The method grants detailed information about a sample by recording the entire spectrum in each pixel of the whole image. We applied HSI to quantify the constituent pH variation in a single infected apoptotic monocyte as a model system. Previously, we showed that the human-pathogenic fungus Aspergillus fumigatus conidia interfere with the acidification of phagolysosomes. Here, we extended this finding to monocytes and gained a more detailed analysis of this process. Our data indicate that melanised A. fumigatus conidia have the ability to interfere with apoptosis in human monocytes as they enable the apoptotic cell to recover from mitochondrial acidification and to continue with the cell cycle. We also showed that this ability of A. fumigatus is dependent on the presence of melanin, since a non-pigmented mutant did not stop the progression of apoptosis and consequently, the cell did not recover from the acidic pH. By conducting the current research based on the HSI, we could measure the intracellular pH in an apoptotic infected human monocyte and show the pattern of pH variation during 35 h of measurements. As a conclusion, we showed the importance of melanin for determining the fate of intracellular pH in a single apoptotic cell. PMID:27727286

  15. Endothelial microparticles (EMP for the assessment of endothelial function: an in vitro and in vivo study on possible interference of plasma lipids.

    Directory of Open Access Journals (Sweden)

    Sabrina H van Ierssel

    Full Text Available BACKGROUND: Circulating endothelial microparticles (EMP reflect the condition of the endothelium and are of increasing interest in cardiovascular and inflammatory diseases. Recently, increased numbers of EMP following oral fat intake, possibly due to acute endothelial injury, have been reported. On the other hand, the direct interference of lipids with the detection of EMP has been suggested. This study aimed to investigate the effect of lipid-rich solutions, commonly administered in clinical practice, on the detection, both in vitro and in vivo, of EMP. METHODS: For the in vitro assessment, several lipid-rich solutions were added to whole blood of healthy subjects (n = 8 and patients with coronary heart disease (n = 5. EMP (CD31+/CD42b- were detected in platelet poor plasma by flow cytometry. For the in vivo study, healthy volunteers were evaluated on 3 different study-days: baseline evaluation, following lipid infusion and after a NaCl infusion. EMP quantification, lipid measurements and peripheral arterial tonometry were performed on each day. RESULTS: Both in vitro addition and in vivo administration of lipids significantly decreased EMP (from 198.6 to 53.0 and from 272.6 to 90.6/µl PPP, respectively, p = 0.001 and p = 0.012. The EMP number correlated inversely with the concentration of triglycerides, both in vitro and in vivo (r = -0.707 and -0.589, p<0.001 and p = 0.021, respectively. The validity of EMP as a marker of endothelial function is supported by their inverse relationship with the reactive hyperemia index (r = -0.758, p = 0.011. This inverse relation was confounded by the intravenous administration of lipids. CONCLUSION: The confounding effect of high circulating levels of lipids, commonly found in patients that receive intravenous lipid-based solutions, should be taken into account when flow cytometry is used to quantify EMP.

  16. In Vitro and In Vivo Differences in Murine Third Complement Component (C3) Opsonization and Macrophage/Leukocyte Responses to Antibody-Functionalized Iron Oxide Nanoworms

    Science.gov (United States)

    Wang, Guankui; Griffin, James I.; Inturi, Swetha; Brenneman, Barbara; Banda, Nirmal K.; Holers, V. Michael; Moghimi, Seyed Moein; Simberg, Dmitri

    2017-01-01

    Balancing surface functionalization and low immune recognition of nanomedicines is a major challenge. Opsonization with the third component of the complement protein (C3) plays a major role in immune cell recognition of nanomedicines. We used dextran-coated superparamagnetic iron oxide nanoworms (SPIO NWs) to study the effect of surface functionalization on C3 opsonization in mouse serum and subsequent macrophage/leukocyte recognition in vitro as well as on intravenous injection into mice. Previously, we found that in mouse serum, SPIO NWs became opsonized with C3 via complement lectin pathway. Crosslinking the dextran shell with epichlorohydrin significantly decreased C3 opsonization and uptake by mouse peritoneal macrophages. Crosslinked nanoworms (NWs) further functionalized with polyethylene glycol (PEG) or with PEG-antibody (Ab) (~160 IgG molecules/particle) did not show an increase in C3 opsonization and peritoneal macrophage uptake in vitro. Following tail vein injection into mice, plain crosslinked NWs and PEGylated crosslinked NWs showed very low C3 opsonization and mouse leukocyte uptake. However, Ab-decorated crosslinked NWs showed significant C3 opsonization and high level of complement-dependent uptake by leukocytes in mice. Decreasing the number of conjugated Abs to 46 IgG molecules/particle significantly reduced C3 opsonization and leukocyte uptake. Using fresh mouse lepirudin plasma rather than serum showed better correlation with C3 opsonization in vivo. The reason for this difference could be related to the known instability of complement classical pathway in mouse sera. Our data illustrate that fine-tuning in nanoparticle surface functionalization with Abs is required to avoid excessive complement activation and complement-mediated immune uptake in mice, and raise issues with in vitro immunological assays of nanomedicines intended to mimic in vivo conditions. PMID:28239384

  17. Dietary Apigenin Exerts Immune-Regulatory Activity in Vivo by Reducing NF-κB Activity, Halting Leukocyte Infiltration and Restoring Normal Metabolic Function

    Science.gov (United States)

    Cardenas, Horacio; Arango, Daniel; Nicholas, Courtney; Duarte, Silvia; Nuovo, Gerard J.; He, Wei; Voss, Oliver H.; Gonzalez-Mejia, M. Elba; Guttridge, Denis C.; Grotewold, Erich; Doseff, Andrea I.

    2016-01-01

    The increasing prevalence of inflammatory diseases and the adverse effects associated with the long-term use of current anti-inflammatory therapies prompt the identification of alternative approaches to reestablish immune balance. Apigenin, an abundant dietary flavonoid, is emerging as a potential regulator of inflammation. Here, we show that apigenin has immune-regulatory activity in vivo. Apigenin conferred survival to mice treated with a lethal dose of Lipopolysaccharide (LPS) restoring normal cardiac function and heart mitochondrial Complex I activity. Despite the adverse effects associated with high levels of splenocyte apoptosis in septic models, apigenin had no effect on reducing cell death. However, we found that apigenin decreased LPS-induced apoptosis in lungs, infiltration of inflammatory cells and chemotactic factors’ accumulation, re-establishing normal lung architecture. Using NF-κB luciferase transgenic mice, we found that apigenin effectively modulated NF-κB activity in the lungs, suggesting the ability of dietary compounds to exert immune-regulatory activity in an organ-specific manner. Collectively, these findings provide novel insights into the underlying immune-regulatory mechanisms of dietary nutraceuticals in vivo. PMID:26938530

  18. Post-ischaemic angiogenic therapy using in vivo prevascularized ascorbic acid-enriched myocardial artificial grafts improves heart function in a rat model.

    Science.gov (United States)

    Martinez, Eliana C; Wang, Jing; Lilyanna, Shera; Ling, Lieng H; Gan, Shu U; Singh, Rajeev; Lee, Chuen N; Kofidis, Theo

    2013-03-01

    Angiogenesis plays a key role in post-ischaemic myocardial repair. We hypothesized that epicardial implantation of an ascorbic acid (AA)-enriched myocardial artificial graft (MAG), which has been prevascularized in the recipients' own body, promotes restoration of the ischaemic heart. Gelatin patches were seeded with GFP-luciferase-expressing rat cardiomyoblasts and enriched with 5 μm AA. Grafts were prevascularized in vivo for 3 days, using a renal pouch model in rats. The MAG patch was then implanted into the same rat's ischaemic heart following myocardial infarction (MI). MAG-treated animals (MAG group, n = 6) were compared to untreated infarcted animals as injury controls (MI group, n = 6) and sham-operated rats as healthy controls (healthy group, n = 7). In vivo bioluminescence imaging indicated a decrease in donor cell survival by 83% during the first week post-implantation. Echocardiographic and haemodynamic assessment 4 weeks after MI revealed that MAG treatment attenuated left ventricular (LV) remodelling (LV end-systolic volume, 0.31 ± 0.13 vs 0.81 ± 0.01 ml, p hearts, attenuated LV remodelling and preserved LV function.

  19. Dietary Apigenin Exerts Immune-Regulatory Activity in Vivo by Reducing NF-κB Activity, Halting Leukocyte Infiltration and Restoring Normal Metabolic Function.

    Science.gov (United States)

    Cardenas, Horacio; Arango, Daniel; Nicholas, Courtney; Duarte, Silvia; Nuovo, Gerard J; He, Wei; Voss, Oliver H; Gonzalez-Mejia, M Elba; Guttridge, Denis C; Grotewold, Erich; Doseff, Andrea I

    2016-03-01

    The increasing prevalence of inflammatory diseases and the adverse effects associated with the long-term use of current anti-inflammatory therapies prompt the identification of alternative approaches to reestablish immune balance. Apigenin, an abundant dietary flavonoid, is emerging as a potential regulator of inflammation. Here, we show that apigenin has immune-regulatory activity in vivo. Apigenin conferred survival to mice treated with a lethal dose of Lipopolysaccharide (LPS) restoring normal cardiac function and heart mitochondrial Complex I activity. Despite the adverse effects associated with high levels of splenocyte apoptosis in septic models, apigenin had no effect on reducing cell death. However, we found that apigenin decreased LPS-induced apoptosis in lungs, infiltration of inflammatory cells and chemotactic factors' accumulation, re-establishing normal lung architecture. Using NF-κB luciferase transgenic mice, we found that apigenin effectively modulated NF-κB activity in the lungs, suggesting the ability of dietary compounds to exert immune-regulatory activity in an organ-specific manner. Collectively, these findings provide novel insights into the underlying immune-regulatory mechanisms of dietary nutraceuticals in vivo.

  20. In vitro and in vivo characterisation of anti-murine IL-13 antibodies recognising distinct functional epitopes.

    Science.gov (United States)

    Berry, L M; Adams, R; Airey, M; Bracher, M G; Bourne, T; Carrington, B; Cross, A S; Davies, G C G; Finney, H M; Foulkes, R; Gozzard, N; Griffin, R A; Hailu, H; Lamour, S D; Lawson, A D; Lightwood, D J; McKnight, A J; O'Dowd, V L; Oxbrow, A K F; Popplewell, A G; Shaw, S; Stephens, P E; Sweeney, B; Tomlinson, K L; Uhe, C; Palframan, R T

    2009-02-01

    Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.

  1. A seven-year storage report of good manufacturing practice-grade naked plasmid DNA: stability, topology, and in vitro/in vivo functional analysis.

    Science.gov (United States)

    Walther, Wolfgang; Schmeer, Marco; Kobelt, Dennis; Baier, Ruth; Harder, Alexander; Walhorn, Volker; Anselmetti, Dario; Aumann, Jutta; Fichtner, Iduna; Schleef, Martin

    2013-12-01

    The great interest for naked plasmid DNA in gene therapy studies is reflected by the fact that it is currently used in 18% of all gene therapy trials. Therefore, validation of topology and functionality of DNA resulting from its long-term stability is an essential requirement for safe and effective gene transfer. To this aim, we analyzed the stability of good manufacturing practice-grade pCMVβ reporter plasmid DNA by capillary gel electrophoresis, agarose gel electrophoresis, and atomic force microscopy. The plasmid DNA was produced for a clinical gene transfer study started in 2005 and was stored for meanwhile 7 years under continuously monitored conditions at -20 °C. The stability of plasmid DNA was monitored by LacZ transgene expression functional assays performed in vitro and in vivo on the 7-year-old plasmid DNA samples compared with plasmid batches newly produced in similar experimental conditions and quality standards. The analyses revealed that during the overall storage time and conditions, the proportion of open circular and supercoiled or covalently closed circular forms is conserved without linearization or degradation of the plasmid. The in vitro transfection and the in vivo jet-injection of DNA showed unaltered functionality of the long-stored plasmid. In summary, the 7-year-old and the newly produced plasmid samples showed similar topology and expression performance. Therefore, our stable storage conditions are effective to preserve the integrity of the DNA to be used in clinical studies. This is an important prerequisite for the long-term performance of gene transfer materials used in trials of long duration as well as of the reference material used in standardization procedures and assays.

  2. A peptide targeting an interaction interface disrupts the dopamine D1-D2 receptor heteromer to block signaling and function in vitro and in vivo: effective selective antagonism

    Science.gov (United States)

    Hasbi, Ahmed; Perreault, Melissa L.; Shen, Maurice Y. F.; Zhang, Lucia; To, Ryan; Fan, Theresa; Nguyen, Tuan; Ji, Xiaodong; O'Dowd, Brian F.; George, Susan R.

    2014-01-01

    Although the dopamine D1-D2 receptor heteromer has emerging physiological relevance and a postulated role in different neuropsychiatric disorders, such as drug addiction, depression, and schizophrenia, there is a need for pharmacological tools that selectively target such receptor complexes in order to analyze their biological and pathophysiological functions. Since no selective antagonists for the D1-D2 heteromer are available, serial deletions and point mutations were used to precisely identify the amino acids involved in an interaction interface between the receptors, residing within the carboxyl tail of the D1 receptor that interacted with the D2 receptor to form the D1-D2 receptor heteromer. It was determined that D1 receptor carboxyl tail residues 404Glu and 405Glu were critical in mediating the interaction with the D2 receptor. Isolated mutation of these residues in the D1 receptor resulted in the loss of agonist activation of the calcium signaling pathway mediated through the D1-D2 receptor heteromer. The physical interaction between the D1 and D2 receptor could be disrupted, as shown by coimmunoprecipitation and BRET analysis, by a small peptide generated from the D1 receptor sequence that contained these amino acids, leading to a switch in G-protein affinities and loss of calcium signaling, resulting in the inhibition of D1-D2 heteromer function. The use of the D1-D2 heteromer-disrupting peptide in vivo revealed a pathophysiological role for the D1-D2 heteromer in the modulation of behavioral despair. This peptide may represent a novel pharmacological tool with potential therapeutic benefits in depression treatment.—Hasbi, A., Perreault, M. L., Shen, M. Y. F., Zhang, L., To, R., Fan, T., Nguyen, T., Ji, X., O'Dowd, B. F., George, S. R. A peptide targeting an interaction interface disrupts the dopamine D1-D2 receptor heteromer to block signaling and function in vitro and in vivo: effective selective antagonism. PMID:25063849

  3. A genetic system to assess in vivo the functions of histones and histone modifications in higher eukaryotes.

    Science.gov (United States)

    Günesdogan, Ufuk; Jäckle, Herbert; Herzig, Alf

    2010-10-01

    Despite the fundamental role of canonical histones in nucleosome structure, there is no experimental system for higher eukaryotes in which basic questions about histone function can be directly addressed. We developed a new genetic tool for Drosophila melanogaster in which the canonical histone complement can be replaced with multiple copies of experimentally modified histone transgenes. This new histone-replacement system provides a well-defined and direct cellular assay system for histone function with which to critically test models in chromatin biology dealing with chromatin assembly, variant histone functions and the biological significance of distinct histone modifications in a multicellular organism.

  4. Special conference of the American Association for Cancer Research on molecular imaging in cancer: linking biology, function, and clinical applications in vivo.

    Science.gov (United States)

    Luker, Gary D

    2002-04-01

    The AACR Special Conference on Molecular Imaging in Cancer: Linking Biology, Function, and Clinical Applications In Vivo, was held January 23-27, 2002, at the Contemporary Hotel, Walt Disney World, Orlando, FL. Co-Chairs David Piwnica-Worms, Patricia Price and Thomas Meade brought together researchers with diverse expertise in molecular biology, gene therapy, chemistry, engineering, pharmacology, and imaging to accelerate progress in developing and applying technologies for imaging specific cellular and molecular signals in living animals and humans. The format of the conference was the presentation of research that focused on basic and translational biology of cancer and current state-of-the-art techniques for molecular imaging in animal models and humans. This report summarizes the special conference on molecular imaging, highlighting the interfaces of molecular biology with animal models, instrumentation, chemistry, and pharmacology that are essential to convert the dreams and promise of molecular imaging into improved understanding, diagnosis, and management of cancer.

  5. In vivo transfection of manganese superoxide dismutase gene or nuclear factor κB shRNA in nodose ganglia improves aortic baroreceptor function in heart failure rats.

    Science.gov (United States)

    Zhang, Dongze; Liu, Jinxu; Tu, Huiyin; Muelleman, Robert L; Cornish, Kurtis G; Li, Yu-Long

    2014-01-01

    Arterial baroreflex sensitivity is attenuated in chronic heart failure (CHF) state, which is associated with cardiac arrhythmias and sudden cardiac death in patients with CHF. Our previous study showed that CHF-induced sodium channel dysfunction in the baroreceptor neurons was involved in the blunted baroreflex sensitivity in CHF rats. Mitochondria-derived superoxide overproduction decreased expression and activation of the sodium channels in the baroreceptor neurons from CHF rats. However, the molecular mechanisms responsible for the sodium channel dysfunction in the baroreceptor neurons from CHF rats remain unknown. We tested the involvement of nuclear factor κB (NFκB) in the sodium channel dysfunction and evaluated the effects of in vivo transfection of manganese superoxide dismutase gene and NFκB shRNA on the baroreflex function in CHF rats. CHF was developed at 6 to 8 weeks after left coronary artery ligation in adult rats. Western blot and chromatin immunoprecipitation data showed that phosphorylated NFκB p65 and ability of NFκB p65 binding to the sodium channel promoter were increased in the nodose ganglia from CHF rats. In vivo transfection of adenoviral manganese superoxide dismutase gene or lentiviral NFκB p65 shRNA into the nodose ganglia partially reversed CHF-reduced sodium channel expression and cell excitability in the baroreceptor neurons and improved CHF-blunted arterial baroreflex sensitivity. Additionally, transfection of adenoviral manganese superoxide dismutase also inhibited the augmentation of phosphorylated NFκB p65 in the nodose neurons from CHF rats. The present study suggests that superoxide-NFκB signaling contributes to CHF-induced baroreceptor dysfunction and resultant impairment of baroreflex function.

  6. N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3′ Splice Site Function in Vivo

    Science.gov (United States)

    Itani, Omar A.; Flibotte, Stephane; Dumas, Kathleen J.; Guo, Chunfang; Blumenthal, Thomas; Hu, Patrick J.

    2016-01-01

    Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3′ splice site. In the nematode Caenorhabditis elegans, 3′ splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3′ splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the –5 position of the octamer motif is the most uncommon base in C. elegans 3′ splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved –5 uridine residue in the C. elegans 3′ splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo. PMID:27172199

  7. Effect of low-temperature ethylene oxide and electron beam sterilization on the in vitro and in vivo function of reconstituted extracellular matrix-derived scaffolds.

    Science.gov (United States)

    Proffen, Benedikt L; Perrone, Gabriel S; Fleming, Braden C; Sieker, Jakob T; Kramer, Joshua; Hawes, Michael L; Murray, Martha M

    2015-10-01

    Reconstituted extracellular matrix (ECM)-derived scaffolds are commonly utilized in preclinical tissue engineering studies as delivery vehicles for cells and growth factors. Translation into clinical use requires identifying a sterilization method that effectively removes bacteria but does not harm scaffold function. To determine effectiveness of sterilization and impact on ECM scaffold integrity and function, low-temperature ethylene oxide and 15 kGy electron beam irradiation techniques were evaluated. Scaffold sterility was assessed in accordance to United States Pharmacopeia Chapter 71. Scaffold matrix degradation was determined in vitro using enzymatic resistance tests and gel electrophoresis. Scaffold mechanics including elastic modulus, yield stress and collapse modulus were tested. Lastly, 14 Yorkshire pigs underwent ACL transection and bio-enhanced ACL repair using sterilized scaffolds. Histologic response of ligament, synovium, and lymph nodes was compared at 4, 6, and 8 weeks. Ethylene oxide as well as electron beam irradiation yielded sterile scaffolds. Scaffold resistance to enzymatic digestion and protein integrity slightly decreased after electron beam irradiation while ethylene oxide altered scaffold matrix. Scaffold elastic modulus and yield stress were increased after electron beam treatment, while collapse modulus was increased after ethylene oxide treatment. No significant changes in ACL dimensions, in vivo scaffold resorption rate, or histologic response of synovium, ligament, and lymph nodes with either terminal sterilization technique were detectable. In conclusion, this study identifies two methods to terminally sterilize an ECM scaffold. In vitro scaffold properties were slightly changed without significantly influencing the biologic responses of the surrounding tissues in vivo. This is a critical step toward translating new tissue engineering strategies to clinical trials. © The Author(s) 2015.

  8. Serotonin transporter function, but not expression, is dependent on brain-derived neurotrophic factor (BDNF): in vivo studies in BDNF-deficient mice.

    Science.gov (United States)

    Daws, L C; Munn, J L; Valdez, M F; Frosto-Burke, T; Hensler, J G

    2007-05-01

    In the present study, we used high-speed chronoamperometry to examine serotonin (5-HT) transporter (5-HTT) function in vivo in 2-, 5-, and 10-month-old brain-derived neurotrophic factor (BDNF)+/- mice. The rate of clearance of exogenously applied 5-HT was measured in CA3 region of hippocampus. In 2-month-old mice, the rate of 5-HT clearance did not differ between BDNF+/+ and BDNF+/- mice. In BDNF+/+ mice, 5-HT clearance rate (Tc) increased markedly with age. In contrast, Tc remained relatively static in BDNF+/- mice across 2-, 5-, and 10-month age groups. At 5 months of age, female BDNF+/+ mice had a lower maximal velocity (Vmax) for 5-HT clearance than male BDNF+/+ mice. There was a similar trend in 5-month-old BDNF+/- mice, but this did not reach statistical significance. There was an age-dependent increase in KT value for 5-HT clearance (i.e., decreased in vivo affinity of 5-HTT), but no significant effect of genotype or gender. 5-HTT density, as measured by [3H]cyanoimipramine binding, was not different between BDNF+/+ and BDNF+/- mice, although there was a significant increase in 5-HTT binding with age. The selective 5-HT reuptake inhibitor fluvoxamine (50 and 100 pmol) significantly decreased 5-HT clearance in BDNF+/+ mice, but not in BDNF+/- mice. Our data suggest that the profoundly reduced ability of 5- and 10-month-old BDNF+/- mice to clear 5-HT is not because of a decrease in the total number of 5-HTTs, but may be due to functional deficits in the 5-HTT, e.g., in the machinery/signaling required for insertion of 5-HTTs into the plasma membrane and/or activation of the 5-HTT once it is positioned to take up 5-HT from extracellular fluid.

  9. Peptide-functionalized starPEG/heparin Hydrogels Direct Mitogenicity, Cell Morphology and Cartilage Matrix Distribution in vitro and in vivo.

    Science.gov (United States)

    Hesse, Eliane; Freudenberg, Uwe; Niemietz, Thomas; Greth, Carina; Weisser, Melanie; Hagmann, Sébastien; Binner, Marcus; Werner, Carsten; Richter, Wiltrud

    2017-01-13

    Cell-based tissue engineering is a promising approach for treating cartilage lesions, but available strategies still provide a distinct composition of the extracellular matrix and an inferior mechanical property compared to native cartilage. To achieve fully functional tissue replacement more rationally designed biomaterials may be needed, introducing bioactive molecules which modulate cell behavior and guide tissue regeneration. This study aimed at exploring the impact of cell instructive, adhesion (GCWGGRGDSP called RGD) and collagen-binding (CKLER/CWYRGRL) peptides, incorporated in a tunable, matrixmetalloprotease (MMP)-responsive multi-arm poly(ethylene glycol) (starPEG)/heparin hydrogel on cartilage regeneration parameters in vitro and in vivo. MMP-responsive-starPEG-conjugates with cysteine termini and heparin-maleimide, optionally pre-functionalized with RGD, CKLER, CWYRGRL or control peptides, were cross-linked by Michael type addition to embed and grow mesenchymal stromal cells (MSC) or chondrocytes. While starPEG/heparin-hydrogel strongly supported chondrogenesis of MSC according to COL2A1, BGN and ACAN induction, MMP-degradability enhanced cell viability and proliferation. RGD-modification of the gels promoted cell spreading with intense cell network formation without negative effects on chondrogenesis. However, CKLER and CWYRGRL were unable to enhance the collagen content of constructs. RGD-modification allowed more even collagen type II distribution by chondrocytes throughout the MMP-responsive constructs especially in vivo. Collectively, peptide-instruction via heparin-enriched MMP-degradable starPEG allowed adjustment of self-renewal, cell morphology and cartilage matrix distribution in order to guide MSC and chondrocyte-based cartilage regeneration towards an improved outcome.

  10. Quantitative assessment of renal function with magnetic resonance imaging; Quantitative Erfassung der renalen Funktion mit der Magnetresonanztomographie

    Energy Technology Data Exchange (ETDEWEB)

    Schoenberg, S.O.; Bock, M.; Aumann, S.; Essig, M.; Floemer, F.; Knopp, M.V.; Kaick, G. van [Deutsches Krebsforschungszentrum Heidelberg (Germany). Abt. Radiologische Diagnostik und Therapie; Just, A. [Heidelberg Univ. (Germany). 1. Physiologisches Inst.

    2000-10-01

    Aim. To show the potential of various methods in magnetic resonance imaging for the evaluation of renal function. Material and Methods. A combined assessment of renal morphology, renal hemodynamics and function is proposed. Various techniques are explained, including multiphasic 3D gadolinium MR angiography, MR phase-contrast flow measurements, quantitative perfusion measurements with intravascular contrast agents, and MR renography and MR urography. The use of these techniques is demonstrated for renovascular diseases. Results. The combined use of these techniques allows renal artery stenosis to be accurately detected and evaluation of renal blood flow, perfusion, glomerular filtration rate, and renal excretion. Based on true quantitative parameters, the hemodynamic and functional significance of the stenosis can be assessed. Renovascular diseases can be differentiated from renoparenchymal disease. Conclusion. For the assessment of renal function, functional magnetic resonance imaging techniques are an important alternative to nuclear medicine. The predictive value regarding the effect of revascularization is currently under investigation. (orig.) [German] Fragestellung. Verschiedene Methoden der Magnetresonanztomographie zur quantitativen Erfassung der Nierenfunktion sollen aufgezeigt werden. Methodik. Es werden MR-Techniken zur kombinierten Beurteilung der renalen Morphologie, Haemodynamik und Funktion der Niere vorgestellt. Zur Anwendung kommen hierbei die multiphasische 3-D-Gadolinium-MR-Angiographie, die MR-Phasenkontrastflussmessung, die quantitative Perfusionsmessung mit intravasalen Kontrastmitteln sowie die MR-Renographie/MR-Urographie. Die Methoden werden exemplarisch am Beispiel renovaskulaerer Erkrankungen demonstriert. Ergebnisse. Mit den vorgestellten Methoden lassen sich Nierenarterienstenosen akkurat detektieren sowie Aenderungen des renalen Blutflusses, der Nierenperfusion, der Filtrationsleistung sowie der renalen Ausscheidung erfassen. Auf der

  11. A three-photon microscope with adaptive optics for deep-tissue in vivo structural and functional brain imaging

    Science.gov (United States)

    Tao, Xiaodong; Lu, Ju; Lam, Tuwin; Rodriguez, Ramiro; Zuo, Yi; Kubby, Joel

    2017-02-01

    We developed a three-photon adaptive optics add-on to a commercial two-photon laser scanning microscope. We demonstrated its capability for structural and functional imaging of neurons labeled with genetically encoded red fluorescent proteins or calcium indicators deep in the living mouse brain with cellular and subcellular resolution.

  12. Fxr1 knockout mice show a striated muscle phenotype: implications for Fxr1p function in vivo.

    NARCIS (Netherlands)

    E.J. Mientjes (Edwin); R. Willemsen (Rob); L.L. Kirkpatrick (Laura); I.M. Nieuwenhuizen (Ingeborg); M. Hoogeveen-Westerveld (Marianne); M. Verweij (Marcel); S. Reis (Surya); B. Bardoni (Barbara); A.T. Hoogeveen (Andre); B.A. Oostra (Ben); D.L. Nelson (David)

    2004-01-01

    textabstractFXR1 is one of the two known homologues of FMR1. FXR1 shares a high degree of sequence homology with FMR1 and also encodes two KH domains and an RGG domain, conferring RNA-binding capabilities. In comparison with FMRP, very little is known about the function of FXR1P in

  13. Blockade of cannabinoid CB receptor function protects against in vivo disseminating brain damage following NMDA-induced excitotoxicity

    DEFF Research Database (Denmark)

    Hansen, H.H.; Ramos, J.A.; Fernández-Ruiz, J.

    2002-01-01

    The ability of cannabinoid CB, receptors to influence glutamatergic excitatory neurotransmission has fueled interest in how these receptors and their endogenous ligands may interact in conditions of excitotoxic insults. The present study characterized the impact of stimulated and inhibited CB...... receptor function on NMDA-induced excitotoxicity. Neonatal (6-day-old) rat pups received a systemic injection of a mixed CB/CB receptor agonist (WIN55,212-2) or their respective antagonists (SR141716A for CB and SR144528 for CB) prior to an unilateral intrastriatal microinjection of NMDA. The NMDA......-induced excitotoxic damage in the ipsilateral forebrain was not influenced by agonist-stimulated CB receptor function. In contrast, blockade of CB, but not CB, receptor activity evoked a robust neuroprotective response by reducing the infarct area and the number of cortical degenerating neurons. These results suggest...

  14. In vivo synthesis of ATPase complexes of Propionigenium modestum and Escherichia coli and analysis of their function.

    Science.gov (United States)

    Gerike, U; Kaim, G; Dimroth, P

    1995-09-01

    Expression studies of Propionigenium modestum ATPase genes in various combinations with Escherichia coli ATPase genes were performed in the unc deletion mutant strain E. coli DK8. Plasmids containing the whole unc operon from P. modestum were unable to complement the E. coli unc deletion mutant. Although all ATPase subunits were expressed from the plasmids, there was no detectable ATP hydrolysing activity, indicating that the F1 part was not functional. Transformants expressing an E. coli F1-P. modestum F0 hybrid exhibited considerable ATPase activities. Binding of the F1 part to the membrane was very weak, however, and the coupling between ATP hydrolysis and Na+ transport was impaired. After combining the genes for E. coli ATPase subunits alpha, beta, gamma, delta and epsilon and the hydrophilic part of subunit b with P. modestum ATPase subunits a and c and the hydrophobic part of subunit b on a plasmid, a non-functional hybrid ATPase was expressed in E. coli. The ATPase was only loosely bound to the membrane, from which it was solubilized with Triton X-100 and purified. Subunit b and a proteolytic degradation product were the only F0 subunits detectable in the purified enzyme. A stable F0 complex is thus not formed with the hybrid b subunit. The absence of a functional F0 complex was in accord with proton-conduction measurements with bacterial vesicles. The only functional Na(+)-translocating ATPase expressed in E. coli thus far consists of E. coli subunits alpha, beta, gamma and epsilon, and P. modestum subunits delta, a, b and c [Kaim, G. & Dimroth, P. (1993) Eur. J. Biochem. 218, 937-944]. During the cloning conducted in our present study, errors in the sequence entry into the EMBL data bank (accession no. X58461) for the P. modestum ATPase alpha and beta subunits became evident, which are corrected in this paper.

  15. Comparing the in vivo function of α-carboxysomes and β-carboxysomes in two model cyanobacteria.

    Science.gov (United States)

    Whitehead, Lynne; Long, Benedict M; Price, G Dean; Badger, Murray R

    2014-05-01

    The carbon dioxide (CO2)-concentrating mechanism of cyanobacteria is characterized by the occurrence of Rubisco-containing microcompartments called carboxysomes within cells. The encapsulation of Rubisco allows for high-CO2 concentrations at the site of fixation, providing an advantage in low-CO2 environments. Cyanobacteria with Form-IA Rubisco contain α-carboxysomes, and cyanobacteria with Form-IB Rubisco contain β-carboxysomes. The two carboxysome types have arisen through convergent evolution, and α-cyanobacteria and β-cyanobacteria occupy different ecological niches. Here, we present, to our knowledge, the first direct comparison of the carboxysome function from α-cyanobacteria (Cyanobium spp. PCC7001) and β-cyanobacteria (Synechococcus spp. PCC7942) with similar inorganic carbon (Ci; as CO2 and HCO3-) transporter systems. Despite evolutionary and structural differences between α-carboxysomes and β-carboxysomes, we found that the two strains are remarkably similar in many physiological parameters, particularly the response of photosynthesis to light and external Ci and their modulation of internal ribulose-1,5-bisphosphate, phosphoglycerate, and Ci pools when grown under comparable conditions. In addition, the different Rubisco forms present in each carboxysome had almost identical kinetic parameters. The conclusions indicate that the possession of different carboxysome types does not significantly influence the physiological function of these species and that similar carboxysome function may be possessed by each carboxysome type. Interestingly, both carboxysome types showed a response to cytosolic Ci, which is of higher affinity than predicted by current models, being saturated by 5 to 15 mm Ci. This finding has bearing on the viability of transplanting functional carboxysomes into the C3 chloroplast.

  16. Human hepatic sinusoidal endothelial cells can be distinguished by expression of phenotypic markers related to their specialised functions in vivo

    Institute of Scientific and Technical Information of China (English)

    PF Lalor; WK Lai; SM Curbishley; S Shetty; DH Adams

    2006-01-01

    The hepatic sinusoids are lined by a unique population of hepatic sinusoidal endothelial cells (HSEC), which is one of the first hepatic cell populations to come into contact with blood components. However, HSEC are not simply barrier cells that restrict the access of bloodborne compounds to the parenchyma. They are functionally specialised endothelial cells that have complex roles, including not only receptor-mediated clearance of endotoxin, bacteria and other compounds, but also the regulation of inflammation, leukocyte recruitment and host immune responses to pathogens. Thus understandlng the differentiation and function of HSEC is critical for the elucidation of liver biology and pathophysiology. This article reviews methods for isolating and studying human hepatic endothelial cell populations using in vitro models. We also discuss the expression and functions of phenotypic markers, such as the presence of fenestrations and expression of VAP-1, Stabilin-1, L-SIGN, which can be used to identify sinusoidal endothelium and to permit discrimination from vascular and lymphatic endothelial cells.

  17. Quantitative evaluation of the reticuloendothelial system function with dynamic MRI.

    Directory of Open Access Journals (Sweden)

    Ting Liu

    Full Text Available PURPOSE: To evaluate the reticuloendothelial system (RES function by real-time imaging blood clearance as well as hepatic uptake of superparamagnetic iron oxide nanoparticle (SPIO using dynamic magnetic resonance imaging (MRI with two-compartment pharmacokinetic modeling. MATERIALS AND METHODS: Kinetics of blood clearance and hepatic accumulation were recorded in young adult male 01b74 athymic nude mice by dynamic T2* weighted MRI after the injection of different doses of SPIO nanoparticles (0.5, 3 or 10 mg Fe/kg. Association parameter, Kin, dissociation parameter, Kout, and elimination constant, Ke, derived from dynamic data with two-compartment model, were used to describe active binding to Kupffer cells and extrahepatic clearance. The clodrosome and liposome were utilized to deplete macrophages and block the RES function to evaluate the capability of the kinetic parameters for investigation of macrophage function and density. RESULTS: The two-compartment model provided a good description for all data and showed a low sum squared residual for all mice (0.27±0.03. A lower Kin, a lower Kout and a lower Ke were found after clodrosome treatment, whereas a lower Kin, a higher Kout and a lower Ke were observed after liposome treatment in comparison to saline treatment (P<0.005. CONCLUSION: Dynamic SPIO-enhanced MR imaging with two-compartment modeling can provide information on RES function on both a cell number and receptor function level.

  18. Direct, quantitative clinical assessment of hand function: usefulness and reproducibility.

    Science.gov (United States)

    Goodson, Alexander; McGregor, Alison H; Douglas, Jane; Taylor, Peter

    2007-05-01

    Methods of assessing functional impairment in arthritic hands include pain assessments and disability scoring scales which are subjective, variable over time and fail to take account of the patients' need to adapt to deformities. The aim of this study was to evaluate measures of functional strength and joint motion in the assessment of the rheumatoid (RA) and osteoarthritic (OA) hand. Ten control subjects, ten RA and ten OA patients were recruited for the study. All underwent pain and disability scoring and functional assessment of the hand using measures of pinch/grip strength and range of joint motion (ROM). Functional assessments including ROM analyses at interphalangeal (IP), metacarpophalangeal (MCP) and wrist joints along with pinch/grip strength clearly discriminated between patient groups (RA vs. OA MCP ROM P<0.0001), pain and disability scales were unable to. In the RA there were demonstrable relationships between ROM measurements and disability (R2=0.31) as well as disease duration (R2=0.37). Intra-patient measures of strength were robust whereas inter-patient comparisons showed variability. In conclusion, pinch/grip strength and ROM are clinically reproducible assessments that may more accurately reflect functional impairment associated with arthritis.

  19. Three Perspectives in Research on Functions: Multi-Representational, Quantitative, and Phenomenological.

    Science.gov (United States)

    Lobato, Joanne; Bowers, Janet

    Much research on student understanding of functions has been characterized by a "multi-representational" perspective that investigates students' efforts to make connections among conventionally accepted mathematical representations such as graphs, tables, and equations. In contrast, a "quantitative" perspective explores…

  20. The effect of muraglitazar on adiponectin signalling, mitochondrial function and fat oxidation genes in human skeletal muscle in vivo