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Sample records for vivo functional analysis

  1. A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)

    DOE PAGES

    Scavuzzo-Duggan, Tess R.; Chaves, Arielle M.; Roberts, Alison W.

    2015-07-14

    Here, a method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Westernmore » blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. In conclusion, compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.« less

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

    PubMed

    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.

  3. A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)

    SciTech Connect

    Scavuzzo-Duggan, Tess R.; Chaves, Arielle M.; Roberts, Alison W.

    2015-07-14

    Here, a method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Western blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. In conclusion, compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.

  4. Functional analysis of articular cartilage deformation, recovery, and fluid flow following dynamic exercise in vivo.

    PubMed

    Eckstein, F; Tieschky, M; Faber, S; Englmeier, K H; Reiser, M

    1999-10-01

    The function of articular cartilage depends on the interaction between the tissue matrix and the interstitial fluid bound to the proteoglycan molecules. Mechanical loading has been shown to be involved in both the metabolic regulation of chondrocytes and in matrix degeneration. The purpose of the present study was therefore to analyze the deformation, recovery, and fluid flow in human articular cartilage after dynamic loading in vivo. The patellae of 7 volunteers were imaged at physical rest and after performing knee bends, with a specifically optimized fat-suppressed FLASH-3D magnetic resonance (MR) sequence. To measure cartilage deformation, the total volume of the patellar cartilage was determined, employing 3D digital image analysis. Patellar cartilage deformation ranged from 2.4 to 8.6% after 50 knee bends, and from 2.4% to 8.5% after 100 knee bends. Repeated sets of dynamic exercise at intervals of 15 min did not cause further deformation. After 100 knee bends, the cartilage required more than 90 min to recover from loading. The rate of fluid flow during relaxation ranged from 1.1 to 3.5 mm(3)/min (0.08 to 0.22 mm(3)/min per square centimeter of the articular surface) and was highly correlated with the individual degree of deformation after knee bends. The data provide the first quantification of articular cartilage recovery and of the rate of fluid flow between the cartilage matrix and surrounding tissue in intact joints in vivo. Measurement in the living opens the possibility of relating interindividual variations of mechanical cartilage properties to the susceptibility of developing joint failure, to assess the load-partitioning between the fluid phase and solid cartilage matrix during load transfer, and to determine the role of mechanically induced fluid flow in the regulation of the metabolic activity of chondrocytes.

  5. Functional analysis of propeptide as an intramolecular chaperone for in vivo folding of subtilisin nattokinase.

    PubMed

    Jia, Yan; Liu, Hui; Bao, Wei; Weng, Meizhi; Chen, Wei; Cai, Yongjun; Zheng, Zhongliang; Zou, Guolin

    2010-12-01

    Here, we show that during in vivo folding of the precursor, the propeptide of subtilisin nattokinase functions as an intramolecular chaperone (IMC) that organises the in vivo folding of the subtilisin domain. Two residues belonging to β-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model.

  6. Toward in vivo two-photon analysis of mouse aqueous outflow structure and function.

    PubMed

    Gonzalez, Jose M; Ko, Minhee K; Masedunskas, Andrius; Hong, Young-Kwon; Weigert, Roberto; Tan, James C H

    2016-05-12

    The promise of revolutionary insights into intraocular pressure (IOP) and aqueous humor outflow homeostasis, IOP pathogenesis, and novel therapy offered by engineered mouse models has been hindered by a lack of appropriate tools for studying the aqueous drainage tissues in their original 3-dimensional (3D) environment. Advances in 2-photon excitation fluorescence imaging (TPEF) combined with availability of modalities such as transgenic reporter mice and intravital dyes have placed us on the cusp of unlocking the potential of the mouse model for unearthing insights into aqueous drainage structure and function. Multimodality 2-photon imaging permits high-resolution visualization not only of tissue structural organization but also cells and cellular function. It is possible to dig deeper into understanding the cellular basis of aqueous outflow regulation as the technique integrates analysis of tissue structure, cell biology and physiology in a way that could also lead to fresh insights into human glaucoma. We outline recent novel applications of two-photon imaging to analyze the mouse conventional drainage system in vivo or in whole tissues: (1) collagen second harmonic generation (SHG) identifies the locations of episcleral vessels, intrascleral plexuses, collector channels, and Schlemm's canal in the distal aqueous drainage tract; (2) the prospero homeobox protein 1-green fluorescent protein (GFP) reporter helps locate the inner wall of Schlemm's canal; (3) Calcein AM, siGLO™, the fluorescent reporters m-Tomato and GFP, and coherent anti-Stokes scattering (CARS), are adjuncts to TPEF to identify live cells by their membrane or cytosolic locations; (4) autofluorescence and sulforhodamine-B to identify elastic fibers in the living eye. These tools greatly expand our options for analyzing physiological and pathological processes in the aqueous drainage tissues of live mice as a model of the analogous human system.

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

    NASA Astrophysics Data System (ADS)

    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

  8. The necessity for in vivo functional analysis in human medical genetics

    PubMed Central

    Quintana, Anita M.

    2015-01-01

    Approximately 50% of all congenital anomalies cannot be linked to any specific genetic etiology, but in recent years cost effective high throughput sequencing has emerged as an efficient strategy for identifying single nucleotide polymorphisms (SNPs) associated with disease. However, in many cases there is not enough evidence to determine if these SNPs underlie disease. To bridge this gap in our understanding advances in functional analyses are warranted. Several preclinical model systems are currently being utilized to provide such evidence, including the advantageous zebrafish embryo. While every system exhibits disadvantages and caveats, a new era of multidisciplinary research has evolved, which uses a broad spectrum of functional analysis tools. This approach will make it possible to identify potential therapeutic targets for both common and rare human disorders. PMID:26989768

  9. The necessity for in vivo functional analysis in human medical genetics.

    PubMed

    Quintana, Anita M

    2015-11-01

    Approximately 50% of all congenital anomalies cannot be linked to any specific genetic etiology, but in recent years cost effective high throughput sequencing has emerged as an efficient strategy for identifying single nucleotide polymorphisms (SNPs) associated with disease. However, in many cases there is not enough evidence to determine if these SNPs underlie disease. To bridge this gap in our understanding advances in functional analyses are warranted. Several preclinical model systems are currently being utilized to provide such evidence, including the advantageous zebrafish embryo. While every system exhibits disadvantages and caveats, a new era of multidisciplinary research has evolved, which uses a broad spectrum of functional analysis tools. This approach will make it possible to identify potential therapeutic targets for both common and rare human disorders.

  10. In Vivo Noninvasive Analysis of Human Forearm Muscle Function and Fatigue: Applications to EVA Operations and Training Maneuvers

    NASA Technical Reports Server (NTRS)

    Fotedar, L. K.; Marshburn, T.; Quast, M. J.; Feeback, D. L.

    1999-01-01

    Forearm muscle fatigue is one of the major limiting factors affecting endurance during performance of deep-space extravehicular activity (EVA) by crew members. Magnetic resonance (MR) provides in vivo noninvasive analysis of tissue level metabolism and fluid exchange dynamics in exercised forearm muscles through the monitoring of proton magnetic resonance imaging (MRI) and phosphorus magnetic resonance spectroscopy (P-31-MRS) parameter variations. Using a space glove box and EVA simulation protocols, we conducted a preliminary MRS/MRI study in a small group of human test subjects during submaximal exercise and recovery and following exhaustive exercise. In assessing simulated EVA-related muscle fatigue and function, this pilot study revealed substantial changes in the MR image longitudinal relaxation times (T2) as an indicator of specific muscle activation and proton flux as well as changes in spectral phosphocreatine-to-phosphate (PCr/Pi) levels as a function of tissue bioenergetic potential.

  11. Independent component analysis for the detection of in vivo intrinsic signals from an optical imager of retinal function

    NASA Astrophysics Data System (ADS)

    Barriga, Eduardo S.; Pattichis, Marios; Abramoff, Michael; T'so, Dan; Kwon, Young; Kardon, Randy; Soliz, Peter

    2007-02-01

    To overcome the difficulty in detection of loss of retinal activity, a functional-Retinal Imaging Device (f-RID) was developed. The device, which is based on a modified fundus camera, seeks to detect changes in optical signals that reflect functional changes in the retina. Measured changes in reflectance in response to the visual stimulus are on the order of 0.1% to 1% of the total reflected intensity level, which makes the functional signal difficult to detect by standard methods because it is masked by other physiological signals and by noise. In this paper, we present a new Independent Component Analysis (ICA) algorithm used to analyze the video sequences from a set of experiments with different patterned stimuli from cats and humans. The ICA algorithm with priors (ICA-P) uses information about the stimulation paradigms to increase the signal detection thresholds when compared to traditional ICA algorithms. The results of the analysis show that we can detect signal levels as low as 0.01% of the total reflected intensity. Also, improvement of up to 30dB in signal detection over traditional ICA algorithms is achieved. The study found that in more than 80% of the in-vivo experiments the patterned stimuli effects on the retina can be detected and extracted.

  12. In vivo analysis of Argos structure-function. Sequence requirements for inhibition of the Drosophila epidermal growth factor receptor.

    PubMed

    Howes, R; Wasserman, J D; Freeman, M

    1998-02-13

    The Drosophila Argos protein is the only known extracellular inhibitor of the epidermal growth factor receptor (EGFR). It is structurally related to the activating ligands, in that it is a secreted protein with a single epidermal growth factor (EGF) domain. To understand the mechanism of Argos inhibition, we have investigated which regions of the protein are essential. A series of deletions were made and tested in vivo; furthermore, by analyzing chimeric proteins between Argos and the activating ligand, Spitz (a transforming growth factor-alpha-like factor), we have examined what makes one inhibitory and the other activating. Our results reveal that Argos has structural requirements that differ from all known EGFR activating ligands; domains flanking the EGF domain are essential for its function. We have also defined the important regions of the atypical Argos EGF domain. The extended B-loop is necessary, whereas the C-loop can be replaced with the equivalent Spitz region without substantially affecting Argos function. Comparison of the argos genes from Drosophila melanogaster and the housefly, Musca domestica, supports our structure-function analysis. These studies are a prerequisite for understanding how Argos inhibits the Drosophila EGFR and provide a basis for designing mammalian EGFR inhibitors.

  13. An in vivo analysis of the vestigial gene in Drosophila melanogaster defines the domains required for Vg function.

    PubMed

    MacKay, Julie O; Soanes, Kelly H; Srivastava, Ajay; Simmonds, Andrew; Brook, William J; Bell, John B

    2003-04-01

    Considerable evidence indicates an obligate partnership of the Drosophila melanogaster Vestigial (VG) and Scalloped (SD) proteins within the context of wing development. These two proteins interact physically and a 56-amino-acid motif within VG is necessary and sufficient for this binding. While the importance of this SD-binding domain has been clearly demonstrated both in vitro and in vivo, the remaining portions of VG have not been examined for in vivo function. Herein, additional regions within VG were tested for possible in vivo functions. The results identify two additional domains that must be present for optimal VG function as measured by the loss of ability to rescue vg mutants, to induce ectopic sd expression, and to perform other normal VG functions when they are deleted. An in vivo study such as this one is fundamentally important because it identifies domains of VG that are necessary in the cellular context in which wing development actually occurs. The results also indicate that an additional large portion of VG, outside of these two domains and the SD-binding domain, is dispensable in the execution of these normal VG functions.

  14. Functional analysis of the catalytic subunit of Dictyostelium PKA in vivo.

    PubMed

    Dammann, H; Traincard, F; Anjard, C; van Bemmelen, M X; Reymond, C; Véron, M

    1998-03-01

    The catalytic subunit of the cAMP-dependent protein kinase (PKA) from Dictyostelium discoideum contains several domains, including an unusually long N-terminal extension preceding a highly conserved catalytic core. We transformed the aggregationless PkaC-null strain with several deletion constructs of both domains. Strains transformed with genes expressing catalytically-inactive polypeptides could not rescue development. Cotransformation with constructs encoding the N-terminal extension and the catalytic core, both unable to rescue development by themselves, yielded transformants able to proceed to late development. A 27-amino acid long hydrophobic region, immediately upstream of the catalytic core, was found indispensable for PKA function. A putative role of this sequence in the acquisition of the active conformation of the protein is discussed.

  15. 3D bioprinting of functional human skin: production and in vivo analysis.

    PubMed

    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.

  16. In vivo analysis of trypanosome mitochondrial RNA function by artificial site-specific RNA endonuclease-mediated knockdown.

    PubMed

    Szempruch, Anthony J; Choudhury, Rajarshi; Wang, Zefeng; Hajduk, Stephen L

    2015-10-01

    Trypanosomes possess a unique mitochondrial genome called the kinetoplast DNA (kDNA). Many kDNA genes encode pre-mRNAs that must undergo guide RNA-directed editing. In addition, alternative mRNA editing gives rise to diverse mRNAs and several kDNA genes encode open reading frames of unknown function. To better understand the mechanism of RNA editing and the function of mitochondrial RNAs in trypanosomes, we have developed a reverse genetic approach using artificial site-specific RNA endonucleases (ASREs) to directly silence kDNA-encoded genes. The RNA-binding domain of an ASRE can be programmed to recognize unique 8-nucleotide sequences, allowing the design of ASREs to cleave any target RNA. Utilizing an ASRE containing a mitochondrial localization signal, we targeted the extensively edited mitochondrial mRNA for the subunit A6 of the F0F1 ATP synthase (A6) in the procyclic stage of Trypanosoma brucei. This developmental stage, found in the midgut of the insect vector, relies on mitochondrial oxidative phosphorylation for ATP production with A6 forming the critical proton half channel across the inner mitochondrial membrane. Expression of an A6-targeted ASRE in procyclic trypanosomes resulted in a 50% reduction in A6 mRNA levels after 24 h, a time-dependent decrease in mitochondrial membrane potential (ΔΨm), and growth arrest. Expression of the A6-ASRE, lacking the mitochondrial localization signal, showed no significant growth defect. The development of the A6-ASRE allowed the first in vivo functional analysis of an edited mitochondrial mRNA in T. brucei and provides a critical new tool to study mitochondrial RNA biology in trypanosomes.

  17. In vivo functional analysis of the Drosophila melanogaster nicotinic acetylcholine receptor Dα6 using the insecticide spinosad.

    PubMed

    Somers, Jason; Nguyen, Joseph; Lumb, Chris; Batterham, Phil; Perry, Trent

    2015-09-01

    The vinegar fly, Drosophila melanogaster, has been used to identify and manipulate insecticide resistance genes. The advancement of genome engineering technology and the increasing availability of pest genome sequences has increased the predictive and diagnostic capacity of the Drosophila model. The Drosophila model can be extended to investigate the basic biology of the interaction between insecticides and the proteins they target. Recently we have developed an in vivo system that permits the expression and study of key insecticide targets, the nicotinic acetylcholine receptors (nAChRs), in controlled genetic backgrounds. Here this system is used to study the interaction between the insecticide spinosad and a nAChR subunit, Dα6. Reciprocal chimeric subunits were created from Dα6 and Dα7, a subunit that does not respond to spinosad. Using the in vivo system, the Dα6/Dα7 chimeric subunits were tested for their capacity to respond to spinosad. Only the subunits containing the C-terminal region of Dα6 were able to respond to spinosad, thus confirming the importance this region for spinosad binding. A new incompletely dominant, spinosad resistance mechanism that may evolve in pest species is also examined. First generated using chemical mutagenesis, the Dα6(P146S) mutation was recreated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, the first use of this technology to introduce a resistant mutation into a controlled genetic background. Both alleles present with the same incompletely dominant, spinosad resistance phenotype, proving the P146S replacement to be the causal mutation. The proximity of the P146S mutation to the conserved Cys-loop indicates that it may impair the gating of the receptor. The results of this study enhance the understanding of nAChR structure:function relationships.

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

    PubMed

    Zeitelhofer, Manuel; Adzemovic, Milena Z; Gomez-Cabrero, David; Bergman, Petra; Hochmeister, Sonja; N'diaye, Marie; Paulson, Atul; Ruhrmann, Sabrina; Almgren, Malin; Tegnér, Jesper N; Ekström, Tomas J; Guerreiro-Cacais, André Ortlieb; Jagodic, Maja

    2017-02-28

    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.

  19. Reversal of coenzyme specificity of 2,3-butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis.

    PubMed

    Ehsani, Maryam; Fernández, Maria R; Biosca, Josep A; Dequin, Sylvie

    2009-10-01

    Saccharomyces cerevisiae NAD(H)-dependent 2,3-butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3-butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)-dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu(221), Ile(222), and Ala(223)) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site-directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration.

  20. Murine T cell clones specific for Hymenolepis nana: generation and functional analysis in vivo and in vitro.

    PubMed

    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. Monitoring in vivo function of cortical microglia.

    PubMed

    Brawek, Bianca; Garaschuk, Olga

    2017-03-14

    Microglia, the innate immune cells of the brain, are becoming increasingly recognized as an important player both in the context of physiological brain function and brain pathology. To fulfill their executive functions microglia can modify their morphology, migrate or move their processes in a directed fashion, and modify the intracellular Ca(2+) dynamics leading to modifications in gene expression, phagocytosis, release of cytokines and other inflammation markers, etc. Here we describe the recently developed tools enabling in vivo monitoring of morphology and Ca(2+) signaling of microglia and show how these techniques may be used for examining microglial function in healthy and diseased brain.

  2. First functional analysis of a novel splicing mutation in the B3GALTL gene by an ex vivo approach in Tunisian patients with typical Peters plus syndrome.

    PubMed

    Ben Mahmoud, Afif; Siala, Olfa; Mansour, Riadh Ben; Driss, Fatma; Baklouti-Gargouri, Siwar; Mkaouar-Rebai, Emna; Belguith, Neila; Fakhfakh, Faiza

    2013-12-10

    Peters plus syndrome is a rare recessive autosomal disorder comprising ocular anterior segment dysgenesis, short stature, hand abnormalities and distinctive facial features. It was related only to mutations in the B3GALTL gene in the 13q12.3 region. In this study, we undertook the first functional analysis of a novel c.597-2 A>G splicing mutation within the B3GALTL gene using an ex-vivo approach. The results showed a complete skipping of exon 8 in the B3GALTL cDNA, which altered the open reading frame of the mutant transcript and generated a PTC within exon 9. This finding potentially elicits the nonsense mRNA to degradation by NMD (nonsense-mediated mRNA decay). The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to ex-vivo results. The findings confirmed the key role played by the B3GALTL gene in typical Peters-plus syndromes and the utility of mRNA analysis to understand the primary impacts of this mutation and the phenotype of the disease.

  3. Analysis of Cardiac Myocyte Maturation Using CASAAV, A Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo.

    PubMed

    Guo, Yuxuan; VanDusen, Nathan J; Zhang, Lina; Gu, Weiliang; Sethi, Isha; Guatimosim, Silvia; Ma, Qing; Jardin, Blake D; Ai, Yulan; Zhang, Donghui; Chen, Biyi; Guo, Ang; Yuan, Guo-Cheng; Song, Long-Sheng; Pu, William T

    2017-03-29

    Rationale: Loss-of-function studies in cardiac myocytes (CMs) are currently limited by the need for appropriate conditional knockout alleles. The factors that regulate CM maturation are poorly understood. Prior studies on CM maturation have been confounded by heart dysfunction caused by whole organ gene inactivation. Objective: To develop a new technical platform to rapidly characterize cell-autonomous gene function in postnatal murine CMs and apply it to identify genes that regulate T-tubules, a hallmark of mature cardiac myocytes. Methods and Results: We developed CASAAV (CRISPR/Cas9-AAV9-based somatic mutagenesis), a platform in which AAV9 delivers tandem guide RNAs targeting a gene of interest and cardiac troponin T promoter (cTNT)-driven Cre to Rosa(Cas9GFP/Cas9GFP) neonatal mice. When directed against junctophilin-2 (Jph2), a gene previously implicated in T-tubule maturation, we achieved efficient, rapid, and CM-specific JPH2 depletion. High-dose AAV9 ablated JPH2 in 64% CMs and caused lethal heart failure, whereas low-dose AAV9 ablated JPH2 in 22% CMs and preserved normal heart function. In the context of preserved heart function, CMs lacking JPH2 developed T-tubules that were nearly morphologically normal, indicating that JPH2 does not have a major, cell-autonomous role in T-tubule maturation. However, in hearts with severe dysfunction, both AAV-transduced and non-transduced CMs exhibited T-tubule disruption, which was more severe in the transduced subset. These data indicate that cardiac dysfunction disrupts T-tubule structure, and that JPH2 protects T-tubules in this context. We then used CASAAV to screen 8 additional genes for required, cell-autonomous roles in T-tubule formation. We identified ryanodine receptor 2 (RYR2) as a novel, cell-autonomously required T-tubule maturation factor. Conclusions: CASAAV is a powerful tool to study cell-autonomous gene functions. Genetic mosaics are invaluable to accurately define cell-autonomous gene function. JPH2

  4. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  5. New models for analyzing mast cell functions in vivo.

    PubMed

    Reber, Laurent L; Marichal, Thomas; Galli, Stephen J

    2012-12-01

    In addition to their well-accepted role as critical effector cells in anaphylaxis and other acute IgE-mediated allergic reactions, mast cells (MCs) have been implicated in a wide variety of processes that contribute to disease or help to maintain health. Although some of these roles were first suggested by analyses of MC products or functions in vitro, it is critical to determine whether, and under which circumstances, such potential roles actually can be performed by MCs in vivo. This review discusses recent advances in the development and analysis of mouse models to investigate the roles of MCs and MC-associated products during biological responses in vivo, and comments on some of the similarities and differences in the results obtained with these newer versus older models of MC deficiency.

  6. Functional nucleic acids as in vivo metabolite and ion biosensors.

    PubMed

    Alsaafin, Alaa; McKeague, Maureen

    2017-02-21

    Characterizing the role of metabolites, metals, and proteins is required to understand normal cell function, and ultimately, elucidate the mechanism of disease. Metabolite concentration and transformation results collected from cell lysates or fixed-cells conceal important dynamic information and differences between individual cells that often have profound functional consequences. Functional nucleic acid-based biosensors are emerging tools that are capable of monitoring ions and metabolites in cell populations or whole animals. Functional nucleic acids (FNAs) are a class of biomolecules that can exhibit either ligand binding or enzymatic activity. Unlike their protein analogues or the use of instrument-based analysis, FNA-based biosensors are capable of entering cells without disruption to the cellular environment and can report on the concentration, dynamics, and spatial localization of molecules in cells. Here, we review the types of FNAs that have been used as in vivo biosensors, and how FNAs can be coupled to transduction systems and delivered inside cells. We also provide examples from the literature that demonstrate their impact in practical applications. Finally, we comment on the critical limitations that need to be addressed to enable their use for single-cell dynamic tracking of metabolites and ions in vivo.

  7. In vivo functions of natural killer cells

    SciTech Connect

    Pollack, S.B.

    1983-01-01

    This review focuses on recent experiments in which the natural killed (NK) compartment has been directly manipulated in vivo either by passive transfer of NK-enriched cell populations or by selection depletion of NK cells. These data have provided direct evidence for the role of NK cells in vivo. It is evident that even these experiments have inherent limitations due to the complexity of in vivo interactions. In the aggregate, however, these data build a compelling case for the in vivo activity of NK cells and for their biologic importance. Most of the experiments were carried out in mice. Although there is heterogeneity among NK cells, these studies deal mainly with classical NK cells defined as bone marrow-derived, non-B (Ig/sup -/), non-T (Lyt 1/sup -/2/sup -/) lymphocytes that are nonadherent and bear the NK-associated antigens NK-1 and asialo-GMl. A natural model which has been exploited to study NK cells in the intact host is also discussed.

  8. Simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system.

    PubMed

    Vinberg, Frans; Kefalov, Vladimir

    2015-05-06

    An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

  9. Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants.

    PubMed

    Violante-Mota, Fernando; Tellechea, Edurne; Moran, Jose F; Sarath, Gautam; Arredondo-Peter, Raúl

    2010-01-01

    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 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 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H2O2 was 86-times lower than that of HRP (K(m)=23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H2O2 as electron donor was 2838-times lower than that of HRP (k(cat)/K(m)=15.8 and 44,833 mM(-1) min(-1), respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H2O2 concentration, and that it poorly protects recombinant Escherichia coli from H2O2 stress. These observations indicate that rice Hb1 inefficiently scavenges H2O2 as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.

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

    PubMed

    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.

  11. Resurrection of DNA Function In Vivo from an Extinct Genome

    PubMed Central

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

    2008-01-01

    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. PMID:18493600

  12. Analysis of TFIIA Function In Vivo: Evidence for a Role in TATA-Binding Protein Recruitment and Gene-Specific Activation

    PubMed Central

    Liu, Qing; Gabriel, Scott E.; Roinick, Kelli L.; Ward, Robert D.; Arndt, Karen M.

    1999-01-01

    Activation of transcription can occur by the facilitated recruitment of TFIID to promoters by gene-specific activators. To investigate the role of TFIIA in TFIID recruitment in vivo, we exploited a class of yeast TATA-binding protein (TBP) mutants that is activation and DNA binding defective. We found that co-overexpression of TOA1 and TOA2, the genes that encode yeast TFIIA, overcomes the activation defects caused by the TBP mutants. Using a genetic screen, we isolated a new class of TFIIA mutants and identified three regions on TFIIA that are likely to be involved in TBP recruitment or stabilization of the TBP-TATA complex in vivo. Amino acid replacements in only one of these regions enhance TFIIA-TBP-DNA complex formation in vitro, suggesting that the other regions are involved in regulatory interactions. To determine the relative importance of TFIIA in the regulation of different genes, we constructed yeast strains to conditionally deplete TFIIA levels prior to gene activation. While the activation of certain genes, such as INO1, was dramatically impaired by TFIIA depletion, activation of other genes, such as CUP1, was unaffected. These data suggest that TFIIA facilitates DNA binding by TBP in vivo, that TFIIA may be regulated by factors that target distinct regions of the protein, and that promoters vary significantly in the degree to which they require TFIIA for activation. PMID:10567590

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

    PubMed

    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.

  14. Functional analysis of the promoter region of amphioxus β-actin gene: a useful tool for driving gene expression in vivo.

    PubMed

    Feng, Jun; Li, Guang; Liu, Xin; Wang, Jing; Wang, Yi-Quan

    2014-10-01

    Amphioxus is a promising new animal model for developmental biology. To develop molecular tools for this model, we characterized the promoter region of a cytoplasmic β-actin gene (Bb-actin-6-2) from the Chinese amphioxus Branchiostoma belcheri. In situ hybridization and real time-quantitative PCR analyses showed that this gene is expressed in many tissues throughout embryonic development. Cloning of cDNA revealed two isoforms with distinct transcription start sites. Isoform #1 exhibits a similar exon/intron and regulatory element organization to that of vertebrate β-actin, whereas isoform #2 lacks the first exon of isoform #1 and recruits its first intron as a promoter. The activities of upstream promoter regions in the two isoforms were examined using the lacZ reporter system in amphioxus embryos. The proximal promoter of isoform #1 drove reporter gene expression broadly in 58.6 % of injected embryos. That of isoform #2 exhibited much higher activity (91.5 %) than that of isoform #1 or the human EF-1-α gene (38.2 %). We determined the minimal promoter regions of the two isoforms via functional analysis. These two regions, alone or inserted a random DNA fragment upstream, had no detectable activity, but when an upstream enhancer was inserted, the promoters directed reporter gene expression in 61.0 and 93.8 %, respectively, of injected embryos in a tissue-specific manner. Our study not only provides insight into the regulatory mechanism underlying amphioxus Bb-actin-6-2 gene expression, but also identifies two sets of efficient proximal and minimal promoters. These promoters could be used to construct gene expression vectors for transgenic studies using amphioxus as a model.

  15. In vivo analysis of Pim-1 deficiency.

    PubMed Central

    Laird, P W; van der Lugt, N M; Clarke, A; Domen, J; Linders, K; McWhir, J; Berns, A; Hooper, M

    1993-01-01

    The Pim-1 proto-oncogene encodes a highly conserved serine/threonine phosphokinase which is predominantly expressed in hematopoietic organs and gonads in mammals. Overexpression of Pim-1 predisposes to lymphomagenesis in mice. To develop a further understanding of Pim-1 in molecular terms, as well as in terms of its potential role in hematopoietic development, we have generated mice deficient in Pim-1 function. Pim-1-deficient mice are ostensibly normal, healthy and fertile. Detailed comparative analyses of the hematopoietic systems of the mutant mice and their wild-type littermates showed that they are indistinguishable for most of the parameters studied. Our analyses revealed one unexpected phenotype that correlated with the level of Pim-1 expression: Pim-1 deficiency correlated with a erythrocyte microcytosis, whereas overexpression of Pim-1 in E mu-Pim-1-transgenic mice resulted in erythrocyte macrocytosis. In order to confirm that the observed decrease in erythrocyte Mean Cell Volume (MCV) was attributable to the Pim-1 deficiency, we developed mice transgenic for a Pim-1 gene construct with its own promoter and showed that this transgene could restore the low erythrocyte Mean Cell Volume observed in the Pim-1-deficient mice to near wild-type levels. These results might be relevant to the observed involvement of the Pim-1 gene in mouse erythroleukemogenesis. The surprising lack of a readily observed phenotype in the lymphoid compartment of the Pim-1-deficient mice, suggests a heretofore unrecognized degree of in vivo functional redundancy of this highly conserved proto-oncogene. Images PMID:8233823

  16. In Vivo 3D Analysis of Thoracic Kinematics: Changes in Size and Shape During Breathing and Their Implications for Respiratory Function in Recent Humans and Fossil Hominins.

    PubMed

    Bastir, Markus; García-Martínez, Daniel; Torres-Tamayo, Nicole; Sanchis-Gimeno, Juan Alberto; O'Higgins, Paul; Utrilla, Cristina; Torres Sánchez, Isabel; García Río, Francisco

    2017-02-01

    The human ribcage expands and contracts during respiration as a result of the interaction between the morphology of the ribs, the costo-vertebral articulations and respiratory muscles. Variations in these factors are said to produce differences in the kinematics of the upper thorax and the lower thorax, but the extent and nature of any such differences and their functional implications have not yet been quantified. Applying geometric morphometrics we measured 402 three-dimensional (3D) landmarks and semilandmarks of 3D models built from computed tomographic scans of thoraces of 20 healthy adult subjects in maximal forced inspiration (FI) and expiration (FE). We addressed the hypothesis that upper and lower parts of the ribcage differ in kinematics and compared different models of functional compartmentalization. During inspiration the thorax superior to the level of the sixth ribs undergoes antero-posterior expansion that differs significantly from the medio-lateral expansion characteristic of the thorax below this level. This supports previous suggestions for dividing the thorax into a pulmonary and diaphragmatic part. While both compartments differed significantly in mean size and shape during FE and FI the size changes in the lower compartment were significantly larger. Additionally, for the same degree of kinematic shape change, the pulmonary thorax changes less in size than the diaphragmatic thorax. Therefore, variations in the form and function of the diaphragmatic thorax will have a strong impact on respiratory function. This has important implications for interpreting differences in thorax shape in terms of respiratory functional differences within and among recent humans and fossil hominins. Anat Rec, 300:255-264, 2017. © 2016 Wiley Periodicals, Inc.

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

    PubMed Central

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

    2015-01-01

    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 to 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. PMID:25738253

  18. Neurovascular coupling: in vivo optical techniques for functional brain imaging

    PubMed Central

    2013-01-01

    Optical imaging techniques reflect different biochemical processes in the brain, which is closely related with neural activity. Scientists and clinicians employ a variety of optical imaging technologies to visualize and study the relationship between neurons, glial cells and blood vessels. In this paper, we present an overview of the current optical approaches used for the in vivo imaging of neurovascular coupling events in small animal models. These techniques include 2-photon microscopy, laser speckle contrast imaging (LSCI), voltage-sensitive dye imaging (VSDi), functional photoacoustic microscopy (fPAM), functional near-infrared spectroscopy imaging (fNIRS) and multimodal imaging techniques. The basic principles of each technique are described in detail, followed by examples of current applications from cutting-edge studies of cerebral neurovascular coupling functions and metabolic. Moreover, we provide a glimpse of the possible ways in which these techniques might be translated to human studies for clinical investigations of pathophysiology and disease. In vivo optical imaging techniques continue to expand and evolve, allowing us to discover fundamental basis of neurovascular coupling roles in cerebral physiology and pathophysiology. PMID:23631798

  19. Neurovascular coupling: in vivo optical techniques for functional brain imaging.

    PubMed

    Liao, Lun-De; Tsytsarev, Vassiliy; Delgado-Martínez, Ignacio; Li, Meng-Lin; Erzurumlu, Reha; Vipin, Ashwati; Orellana, Josue; Lin, Yan-Ren; Lai, Hsin-Yi; Chen, You-Yin; Thakor, Nitish V

    2013-04-30

    Optical imaging techniques reflect different biochemical processes in the brain, which is closely related with neural activity. Scientists and clinicians employ a variety of optical imaging technologies to visualize and study the relationship between neurons, glial cells and blood vessels. In this paper, we present an overview of the current optical approaches used for the in vivo imaging of neurovascular coupling events in small animal models. These techniques include 2-photon microscopy, laser speckle contrast imaging (LSCI), voltage-sensitive dye imaging (VSDi), functional photoacoustic microscopy (fPAM), functional near-infrared spectroscopy imaging (fNIRS) and multimodal imaging techniques. The basic principles of each technique are described in detail, followed by examples of current applications from cutting-edge studies of cerebral neurovascular coupling functions and metabolic. Moreover, we provide a glimpse of the possible ways in which these techniques might be translated to human studies for clinical investigations of pathophysiology and disease. In vivo optical imaging techniques continue to expand and evolve, allowing us to discover fundamental basis of neurovascular coupling roles in cerebral physiology and pathophysiology.

  20. Posterior lymph heart function in two species of anurans: analysis based on both in vivo pressure-volume relationships by conductance manometry and ultrasound.

    PubMed

    Crossley, Dane A; Hillman, Stanley S

    2010-11-01

    Rhinella marina and Lithobates catesbeianus have known differences in the capacity to mobilize lymph to stabilize blood volume following dehydration and hemorrhage. The purpose of these experiments was to assess whether there are interspecific differences in basic lymph heart functions. The end diastolic volumes of posterior lymph hearts averaged 10.8 μl kg⁻¹ in R. marina and 7.9-10.8 μl kg⁻¹ in L. catesbeianus by conductance manometry, and 9-32 μl kg⁻¹ in R. marina by ultrasound techniques, which correlated with body mass. Stroke volumes were approximately 20% of end diastolic volumes in both species. Peak systolic pressures and stroke work were correlated with the index of contractility (dP/dt(max)) in both species. Stroke volume was correlated to stroke work but not peak systolic pressure, end diastolic volume or end diastolic pressure indicating the preload variables do not seem to determine stroke volume as would be predicted from Starling considerations of the blood heart. Renal portal elastance (end systolic pressure/stroke volume) an afterload index did not differ interspecifically, and was equivalent to values for systemic flow indices from mice of equivalent ventricular volume. These data, taken together with predictions derived from mammalian models on the effect of high resistance indicate afterload (renal portal pressure), may be important determinants of posterior lymph heart stroke volume. The shape of the pressure-volume loop is different from an idealized version previously reported, and is influenced by end diastolic volume. Our data indicate that increasing end diastolic pressure and volume can influence the loop shape but not the stroke volume. This indicates that lymph hearts do not behave in a Starling Law manner with increased preload volume.

  1. In-vivo plasma-mediated ablation as a function of laser pulse width

    NASA Astrophysics Data System (ADS)

    Liu, Xinbing; Tien, An-Chun; Juhasz, Tibor; Irish, Barbara; Elner, Victor; Kurtz, Ron M.

    1997-06-01

    We evaluated in vivo wound healing responses to plasma- mediated ablation in skin as a function of laser pulsewidth and energy. Experiments utilized a regeneratively amplified Ti:Sapphire laser operating at 800 nm with pulsewidths varied from 7 ns to 100 fs. Skin incisions were created in mice by tightly focusing the laser beam on the tissue surface. Incisions of equal depth were compared at time points ranging from 6 hours to 3 weeks using standard histologic methods. Incision depth was proportional to pulse energy at each pulsewidth. Fluence threshold dependence on laser pulsewidth agreed with those predicted by ex vivo testing. Histologic analysis revealed minimal adjacent tissue damage at pulsewidths less than a few picoseconds and energies near the fluence threshold. Longer pulsewidths and higher fluence levels were associated with more significant collateral effects. These in vivo results suggest collateral tissue damage and secondary effects may be minimized by controlling laser pulsewidth and energy.

  2. Functional Extended Redundancy Analysis

    ERIC Educational Resources Information Center

    Hwang, Heungsun; Suk, Hye Won; Lee, Jang-Han; Moskowitz, D. S.; Lim, Jooseop

    2012-01-01

    We propose a functional version of extended redundancy analysis that examines directional relationships among several sets of multivariate variables. As in extended redundancy analysis, the proposed method posits that a weighed composite of each set of exogenous variables influences a set of endogenous variables. It further considers endogenous…

  3. Functional and Molecular Characterization of Ex Vivo Cultured Epiretinal Membrane Cells from Human Proliferative Diabetic Retinopathy

    PubMed Central

    Veréb, Zoltán; Lumi, Xhevat; Andjelic, Sofija; Globocnik-Petrovic, Mojca; Urbancic, Mojca; Hawlina, Marko; Facskó, Andrea; Petrovski, Goran

    2013-01-01

    Characterization of the cell surface marker phenotype of ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherently ex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNFα activation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when cultured ex vivo, which were negative for endothelial cell markers (CD31, VEGFR2), partially positive for hematopoietic- (CD34, CD47) and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFRβ), and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1) were detected upon TNFα activation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDR ex vivo. PMID:24195074

  4. Functional and molecular characterization of ex vivo cultured epiretinal membrane cells from human proliferative diabetic retinopathy.

    PubMed

    Veréb, Zoltán; Lumi, Xhevat; Andjelic, Sofija; Globocnik-Petrovic, Mojca; Urbancic, Mojca; Hawlina, Marko; Facskó, Andrea; Petrovski, Goran

    2013-01-01

    Characterization of the cell surface marker phenotype of ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherently ex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNF α activation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when cultured ex vivo, which were negative for endothelial cell markers (CD31, VEGFR2), partially positive for hematopoietic- (CD34, CD47) and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFR β ), and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1) were detected upon TNF α activation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDR ex vivo.

  5. Functional imaging: monitoring heme oxygenase-1 gene expression in vivo

    NASA Astrophysics Data System (ADS)

    Zhang, Weisheng; Reilly-Contag, Pamela; Stevenson, David K.; Contag, Christopher H.

    1999-07-01

    The regulation of genetic elements can be monitored in living animals using photoproteins as reporters. Heme oxygenase (HO) is the key catabolic enzyme in the heme degradation pathway. Here, HO expression serves as a model for in vivo functional imaging of transcriptional regulation of a clinically relevant gene. HO enzymatic activity is inhibited by heme analogs, metalloporphyrins, but many members of this family of compounds also activate transcription of the HO-1 promoter. The degree of transcriptional activation by twelve metalloporphyrins, differing at the central metal and porphyrin ring substituents, was evaluated in both NIH 3T3 stable lines and transgenic animals containing HO-1 promoter-luciferase gene fusions. In the correlative cell culture assays, the metalloporphyrins increased transcription form the full length HO promoter fusion to varying degrees, but none increased transcription from a truncated HO-1 promoter. These results suggested that one or both of the two distal enhancer elements located at -4 and -10 Kb upstream from transcriptional start are required for HO-1 induction by heme and its analogs. The full-length HO-1-luc fusion was then evaluated as a transgene in mice. It was possible to monitor the effects of the metalloporphyrins, SnMP and ZnPP, in living animals over time. This spatiotemporal analyses of gene expression in vivo implied that alterations in porphyrin ring substituents and the central metal may affect the extent of gene activation. These data further indicate that using photoprotein reporters, subtle differences in gene expression can be monitored in living animals.

  6. Macrophages form functional vascular mimicry channels in vivo

    PubMed Central

    Barnett, Faith H.; Rosenfeld, Mauricio; Wood, Malcolm; Kiosses, William B.; Usui, Yoshihiko; Marchetti, Valentina; Aguilar, Edith; Friedlander, Martin

    2016-01-01

    Macrophages, key cells of the innate immune system, are known to support angiogenesis but are not believed to directly form vessel walls. Here we show that macrophages structurally form primitive, NON-ENDOTHELIAL “vessels” or vascular mimicry (VM) channels in both tumor and angiogenesis in vivo models. These channels are functionally connected to the systemic vasculature as they are perfused by intravenously injected dye. Since both models share hypoxic micro-environments, we hypothesized that hypoxia may be an important mediator of VM formation. Indeed, conditional genetic depletion of myeloid-specific HIF-1α results in decreased VM network formation, dye perfusion and tumor size. Although the macrophage VM network shares some features with an endothelial vasculature, it is ultrastructurally different. Cancer stem cells have been shown to form vascular mimicry channels. Our data demonstrates that tumor-associated macrophages also form them. The identification of this novel type of vascular mimicry may help in the development of targeted cancer therapeutics. PMID:27834402

  7. Assessing in vivo microRNA function in the germline stem cells of the Drosophila ovary.

    PubMed

    Chan, Kin; Ruohola-Baker, Hannele

    2010-01-01

    A more complete understanding of the biology of adult stem cells could yield important insights toward devising effective cell-based regenerative therapies to treat disease. The germline stem cells (GSCs) in the fruit fly Drosophila melanogaster are an excellent in vivo model for the study of adult stem cell biology. There is increasing evidence from a growing field that microRNAs (miRNAs) play important roles in controlling many aspects of stem-cell biology. Using straightforward genetic manipulations combined with well-established cell biological analysis techniques, we and others have found that the miRNA pathway regulates the cell division rate of Drosophila GSCs as well as the maintenance of the GSCs in their niche. In this chapter, we offer a detailed, self-contained description of a general method to assess the in vivo functions of miRNAs in the GSCs of the Drosophila ovary.

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

    SciTech Connect

    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.

  9. Fetal in vivo continuous cardiovascular function during chronic hypoxia

    PubMed Central

    Allison, B. J.; Brain, K. L.; Niu, Y.; Kane, A. D.; Herrera, E. A.; Thakor, A. S.; Botting, K. J.; Cross, C. M.; Itani, N.; Skeffington, K. L.; Beck, C.

    2016-01-01

    Key points The in vivo fetal cardiovascular defence to chronic hypoxia has remained by and large an enigma because no technology has been available to induce significant and prolonged fetal hypoxia whilst recording longitudinal changes in fetal regional blood flow as the hypoxic pregnancy is developing.We introduce a new technique able to maintain chronically instrumented maternal and fetal sheep preparations under isobaric chronic hypoxia for most of gestation, beyond levels that can be achieved by high altitude and of relevance in magnitude to the human intrauterine growth‐restricted fetus.This technology permits wireless recording in free‐moving animals of longitudinal maternal and fetal cardiovascular function, including beat‐to‐beat alterations in pressure and blood flow signals in regional circulations.The relevance and utility of the technique is presented by testing the hypotheses that the fetal circulatory brain sparing response persists during chronic fetal hypoxia and that an increase in reactive oxygen species in the fetal circulation is an involved mechanism. Abstract Although the fetal cardiovascular defence to acute hypoxia and the physiology underlying it have been established for decades, how the fetal cardiovascular system responds to chronic hypoxia has been comparatively understudied. We designed and created isobaric hypoxic chambers able to maintain pregnant sheep for prolonged periods of gestation under controlled significant (10% O2) hypoxia, yielding fetal mean PaO2 levels (11.5 ± 0.6 mmHg) similar to those measured in human fetuses of hypoxic pregnancy. We also created a wireless data acquisition system able to record fetal blood flow signals in addition to fetal blood pressure and heart rate from free moving ewes as the hypoxic pregnancy is developing. We determined in vivo longitudinal changes in fetal cardiovascular function including parallel measurement of fetal carotid and femoral blood flow and oxygen and glucose delivery

  10. Functional group analysis

    SciTech Connect

    Smith, W.T. Jr.; Patterson, J.M.

    1986-04-01

    Analytical methods for functional group analysis are reviewed. Literature reviewed is from the period of December 1983 through November 1985 and presents methods for determining the following compounds: acids, acid halides, active hydrogen, alcohols, aldehydes, ketones, amides, amines, amino acids, anhydrides, aromatic hydrocarbons, azo compounds, carbohydrates, chloramines, esters, ethers, halogen compounds, hydrazines, isothiocyanates, nitro compounds, nitroso compounds, organometallic compounds, oxiranes, peroxides, phenols, phosphorus compounds, quinones, silicon compounds, sulfates, sulfonyl chlorides, thioamides, thiols, and thiosemicarbazones. 150 references.

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

    PubMed Central

    Jiang, Xue; Chen, Yuxi; Zhang, Zhen; Zhang, Xiya; Liang, Puping; Zhan, Shaoquan; Cao, Shanbo; Songyang, Zhou; Huang, Junjiu

    2015-01-01

    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. PMID:26599493

  12. In vivo analysis of polyadenylation in prokaryotes.

    PubMed

    Mohanty, Bijoy K; Kushner, Sidney R

    2014-01-01

    Polyadenylation at the 3' ends of mRNAs, tRNAs, rRNAs, and sRNAs plays important roles in RNA metabolism in both prokaryotes and eukaryotes. However, the nature of poly(A) tails in prokaryotes is distinct compared to their eukaryotic counterparts. Specifically, depending on the organism, eukaryotic poly(A) tails average between 50 and >200 nt and can easily be isolated by several techniques involving oligo(dT)-dependent cDNA amplification. In contrast, the bulk of the poly(A) tails present on prokaryotic transcripts is relatively short (<10 nt) and is difficult to characterize using similar techniques. This chapter describes methods that can circumvent these problems. For example, we discuss how to isolate total RNA and characterize its overall polyadenylation status employing a poly(A) sizing assay. Furthermore, we describe a technique involving RNase H treatment of total RNA followed by northern analysis in order to distinguish length of poly(A) tails on various types of transcripts. Finally, we outline a useful procedure to clone the poly(A) tails of specific transcripts using 5'-3' end-ligated RNA, which is independent of oligo(dT)-dependent cDNA amplification. These approaches are particularly helpful in analyzing transcripts with either short or long poly(A) tails both in prokaryotes and eukaryotes.

  13. Analysis of Cortical Flow Models In Vivo

    PubMed Central

    Benink, Hélène A.; Mandato, Craig A.; Bement, William M.

    2000-01-01

    Cortical flow, the directed movement of cortical F-actin and cortical organelles, is a basic cellular motility process. Microtubules are thought to somehow direct cortical flow, but whether they do so by stimulating or inhibiting contraction of the cortical actin cytoskeleton is the subject of debate. Treatment of Xenopus oocytes with phorbol 12-myristate 13-acetate (PMA) triggers cortical flow toward the animal pole of the oocyte; this flow is suppressed by microtubules. To determine how this suppression occurs and whether it can control the direction of cortical flow, oocytes were subjected to localized manipulation of either the contractile stimulus (PMA) or microtubules. Localized PMA application resulted in redirection of cortical flow toward the site of application, as judged by movement of cortical pigment granules, cortical F-actin, and cortical myosin-2A. Such redirected flow was accelerated by microtubule depolymerization, showing that the suppression of cortical flow by microtubules is independent of the direction of flow. Direct observation of cortical F-actin by time-lapse confocal analysis in combination with photobleaching showed that cortical flow is driven by contraction of the cortical F-actin network and that microtubules suppress this contraction. The oocyte germinal vesicle serves as a microtubule organizing center in Xenopus oocytes; experimental displacement of the germinal vesicle toward the animal pole resulted in localized flow away from the animal pole. The results show that 1) cortical flow is directed toward areas of localized contraction of the cortical F-actin cytoskeleton; 2) microtubules suppress cortical flow by inhibiting contraction of the cortical F-actin cytoskeleton; and 3) localized, microtubule-dependent suppression of actomyosin-based contraction can control the direction of cortical flow. We discuss these findings in light of current models of cortical flow. PMID:10930453

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

    PubMed Central

    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

  15. Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice.

    PubMed

    Gaudenzio, Nicolas; Sibilano, Riccardo; Starkl, Philipp; Tsai, Mindy; Galli, Stephen J; Reber, Laurent L

    2015-05-27

    Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo approaches over in vitro methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the 'mast cell knock-in' approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

  16. Analysis of gamma-aminobutyric acidB receptor function in the in vitro and in vivo regulation of alpha-melanotropin-stimulating hormone secretion from melanotrope cells of Xenopus laevis.

    PubMed

    De Koning, H P; Jenks, B G; Roubos, E W

    1993-02-01

    The activity of many endocrine cells is regulated by gamma-aminobutyric acid (GABA). The effects of GABA are mediated by GABAA and/or GABAB receptors. While GABAB receptors in the central nervous system have now been extensively characterized, little is known of the function and pharmacology of GABAB receptors on endocrine cells. In the amphibian Xenopus laevis, GABA inhibits the release of alpha MSH from the endocrine melanotrope cells through both GABAA and GABAB receptors. We have investigated the following aspects of the GABAB receptor of the melanotrope cells of X. laevis: 1) the pharmacology of this receptor, using antagonists previously established to demonstrate GABAB receptors in the mammalian central nervous system; 2) the relative contribution to the regulation of hormone secretion by the GABAA and GABAB receptors on melanotrope cells in vitro; and 3) the role of the GABAB receptor with respect to the physiological function of the melanotrope cell in vivo, i.e. regulation of pigment dispersion in skin melanophores in relation to background color. Our results demonstrate that phaclofen, 2-hydroxysaclofen, and 4-aminobutylphosphonic acid dose-dependently blocked the inhibition of alpha MSH release by GABAB receptor activation, but not by GABAA receptor activation. The GABAB receptor antagonist delta-aminovaleric acid appeared to be a selective agonist on the GABAB receptor of melanotrope cells. The inhibitory secretory response to a low dose of GABA (10(-5) M) was not affected by bicuculline, but was significantly reduced by phaclofen, indicating that at a low GABA concentration, the GABAB receptor mechanism would dominate in inhibiting the melanotrope cells. Different thresholds of activation may form the basis for differential action of GABA through both GABA receptor types. The tonic inhibition of alpha MSH release in animals adapted to a white background was not affected by 4-aminobutylphosphonic acid, indicating that the GABAB receptor is not (solely

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

    PubMed Central

    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

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

    PubMed

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

    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.

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:27703156

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

    PubMed

    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.

  1. Proteomics meets genetics: SILAC labeling of Drosophila melanogaster larvae and cells for in vivo functional studies.

    PubMed

    Cuomo, Alessandro; Sanfilippo, Roberta; Vaccari, Thomas; Bonaldi, Tiziana

    2014-01-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is an established and potent method for quantitative proteomics. When combined with high-resolution mass spectrometry (MS) and efficient algorithms for the analysis of quantitative MS data, SILAC has proven to be the strategy of choice for the in-depth characterization of functional states at the protein level. The fruit fly Drosophila melanogaster is one of the most widely used model systems for studies of genetics and developmental biology. Despite this, a global proteomic approach in Drosophila is rarely considered. Here, we describe an adaptation of SILAC for functional investigation of fruit flies by proteomics: We illustrate how to perform efficient SILAC labeling of cells in culture and whole fly larvae. The combination of SILAC, a highly accurate global protein quantification method, and of the fruit fly, the prime genetics and developmental model, represents a unique opportunity for quantitative proteomic studies in vivo.

  2. Single-cell analysis of endothelial morphogenesis in vivo.

    PubMed

    Yu, Jianxin A; Castranova, Daniel; Pham, Van N; Weinstein, Brant M

    2015-09-01

    Vessel formation has been extensively studied at the tissue level, but the difficulty in imaging the endothelium with cellular resolution has hampered study of the morphogenesis and behavior of endothelial cells (ECs) in vivo. We are using endothelial-specific transgenes and high-resolution imaging to examine single ECs in zebrafish. By generating mosaics with transgenes that simultaneously mark endothelial nuclei and membranes we are able to definitively identify and study the morphology and behavior of individual ECs during vessel sprouting and lumen formation. Using these methods, we show that developing trunk vessels are composed of ECs of varying morphology, and that single-cell analysis can be used to quantitate alterations in morphology and dynamics in ECs that are defective in proper guidance and patterning. Finally, we use single-cell analysis of intersegmental vessels undergoing lumen formation to demonstrate the coexistence of seamless transcellular lumens and single or multicellular enclosed lumens with autocellular or intercellular junctions, suggesting that heterogeneous mechanisms contribute to vascular lumen formation in vivo. The tools that we have developed for single EC analysis should facilitate further rigorous qualitative and quantitative analysis of EC morphology and behavior in vivo.

  3. In Vivo Imaging Techniques: A New Era for Histochemical Analysis

    PubMed Central

    Busato, A.; Feruglio, P. Fumene; Parnigotto, P.P.; Marzola, P.; Sbarbati, A.

    2016-01-01

    In vivo imaging techniques can be integrated with classical histochemistry to create an actual histochemistry of water. In particular, Magnetic Resonance Imaging (MRI), an imaging technique primarily used as diagnostic tool in clinical/preclinical research, has excellent anatomical resolution, unlimited penetration depth and intrinsic soft tissue contrast. Thanks to the technological development, MRI is not only capable to provide morphological information but also and more interestingly functional, biophysical and molecular. In this paper we describe the main features of several advanced imaging techniques, such as MRI microscopy, Magnetic Resonance Spectroscopy, functional MRI, Diffusion Tensor Imaging and MRI with contrast agent as a useful support to classical histochemistry. PMID:28076937

  4. In Vivo Application of Optogenetics for Neural Circuit Analysis

    PubMed Central

    2012-01-01

    Optogenetics combines optical and genetic methods to rapidly and reversibly control neural activities or other cellular functions. Using genetic methods, specific cells or anatomical pathways can be sensitized to light through exogenous expression of microbial light activated opsin proteins. Using optical methods, opsin expressing cells can be rapidly and reversibly controlled by pulses of light of specific wavelength. With the high spatial temporal precision, optogenetic tools have enabled new ways to probe the causal role of specific cells in neural computation and behavior. Here, we overview the current state of the technology, and provide a brief introduction to the practical considerations in applying optogenetics in vivo to analyze neural circuit functions. PMID:22896801

  5. In vivo characterization of regenerative peripheral nerve interface function

    NASA Astrophysics Data System (ADS)

    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

  6. Functional genetic targeting of embryonic kidney progenitor cells ex vivo.

    PubMed

    Junttila, Sanna; Saarela, Ulla; Halt, Kimmo; Manninen, Aki; Pärssinen, Heikki; Lecca, M Rita; Brändli, André W; Sims-Lucas, Sunder; Skovorodkin, Ilya; Vainio, Seppo J

    2015-05-01

    The embryonic mammalian metanephric mesenchyme (MM) is a unique tissue because it is competent to generate the nephrons in response to Wnt signaling. An ex vivo culture in which the MM is separated from the ureteric bud (UB), the natural inducer, can be used as a classic tubule induction model for studying nephrogenesis. However, technological restrictions currently prevent using this model to study the molecular genetic details before or during tubule induction. Using nephron segment-specific markers, we now show that tubule induction in the MM ex vivo also leads to the assembly of highly segmented nephrons. This induction capacity was reconstituted when MM tissue was dissociated into a cell suspension and then reaggregated (drMM) in the presence of human recombinant bone morphogenetic protein 7/human recombinant fibroblast growth factor 2 for 24 hours before induction. Growth factor-treated drMM also recovered the capacity for organogenesis when recombined with the UB. Cell tracking and time-lapse imaging of chimeric drMM cultures indicated that the nephron is not derived from a single progenitor cell. Furthermore, viral vector-mediated transduction of green fluorescent protein was much more efficient in dissociated MM cells than in intact mesenchyme, and the nephrogenic competence of transduced drMM progenitor cells was preserved. Moreover, drMM cells transduced with viral vectors mediating Lhx1 knockdown were excluded from the nephric tubules, whereas cells transduced with control vectors were incorporated. In summary, these techniques allow reproducible cellular and molecular examinations of the mechanisms behind nephrogenesis and kidney organogenesis in an ex vivo organ culture/organoid setting.

  7. A Primer on Functional Analysis

    ERIC Educational Resources Information Center

    Yoman, Jerome

    2008-01-01

    This article presents principles and basic steps for practitioners to complete a functional analysis of client behavior. The emphasis is on application of functional analysis to adult mental health clients. The article includes a detailed flow chart containing all major functional diagnoses and behavioral interventions, with functional assessment…

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27301303

  11. In vivo platforms for analysis of HIV persistence and eradication.

    PubMed

    Garcia, J Victor

    2016-02-01

    HIV persistence in patients undergoing antiretroviral therapy is a major impediment to the cure of HIV/AIDS. The molecular and cellular mechanisms underlying HIV persistence in vivo have not been fully elucidated. This lack of basic knowledge has hindered progress in this area. The in vivo analysis of HIV persistence and the implementation of curative strategies would benefit from animal models that accurately recapitulate key aspects of the human condition. This Review summarizes the contribution that humanized mouse models of HIV infection have made to the field of HIV cure research. Even though these models have been shown to be highly informative in many specific areas, their great potential to serve as excellent platforms for discovery in HIV pathogenesis and treatment has yet to be fully developed.

  12. In vivo osteogenesis assay: a rapid method for quantitative analysis.

    PubMed

    Dennis, J E; Konstantakos, E K; Arm, D; Caplan, A I

    1998-08-01

    A quantitative in vivo osteogenesis assay is a useful tool for the analysis of cells and bioactive factors that affect the amount or rate of bone formation. There are currently two assays in general use for the in vivo assessment of osteogenesis by isolated cells: diffusion chambers and porous calcium phosphate ceramics. Due to the relative ease of specimen preparation and reproducibility of results, the porous ceramic assay was chosen for the development of a rapid method for quantitating in vivo bone formation. The ceramic cube implantation technique consists of combining osteogenic cells with 27-mm3 porous calcium phosphate ceramics, implanting the cell-ceramic composites subcutaneously into an immuno-tolerant host, and, after 2-6 weeks, harvesting and preparing the ceramic implants for histologic analysis. A drawback to the analysis of bone formation within these porous ceramics is that the entire cube must be examined to find small foci of bone present in some samples; a single cross-sectional area is not representative. For this reason, image analysis of serial sections from ceramics is often prohibitively time-consuming. Two alternative scoring methodologies were tested and compared to bone volume measurements obtained by image analysis. The two subjective scoring methods were: (1) Bone Scale: the amount of bone within pores of the ceramic implant is estimated on a scale of 0-4 based on the degree of bone fill (0=no bone, 1=up to 25%, 2=25 to 75%, 4=75 to 100% fill); and (2) Percentage Bone: the amount of bone is estimated by determining the percentage of ceramic pores which contain bone. Every tenth section of serially sectioned cubes was scored by each of these methods under double-blind conditions, and the Bone Scale and Percentage Bone results were directly compared to image analysis measurements from identical samples. Correlation coefficients indicate that the Percentage Bone method was more accurate than the Bone Scale scoring method. The Bone Scale

  13. Function Point Analysis Depot

    NASA Technical Reports Server (NTRS)

    Muniz, R.; Martinez, El; Szafran, J.; Dalton, A.

    2011-01-01

    The Function Point Analysis (FPA) Depot is a web application originally designed by one of the NE-C3 branch's engineers, Jamie Szafran, and created specifically for the Software Development team of the Launch Control Systems (LCS) project. The application consists of evaluating the work of each developer to be able to get a real estimate of the hours that is going to be assigned to a specific task of development. The Architect Team had made design change requests for the depot to change the schema of the application's information; that information, changed in the database, needed to be changed in the graphical user interface (GUI) (written in Ruby on Rails (RoR and the web service/server side in Java to match the database changes. These changes were made by two interns from NE-C, Ricardo Muniz from NE-C3, who made all the schema changes for the GUI in RoR and Edwin Martinez, from NE-C2, who made all the changes in the Java side.

  14. Understanding functional miRNA-target interactions in vivo by site-specific genome engineering.

    PubMed

    Bassett, Andrew R; Azzam, Ghows; Wheatley, Lucy; Tibbit, Charlotte; Rajakumar, Timothy; McGowan, Simon; Stanger, Nathan; Ewels, Philip Andrew; Taylor, Stephen; Ponting, Chris P; Liu, Ji-Long; Sauka-Spengler, Tatjana; Fulga, Tudor A

    2014-08-19

    MicroRNA (miRNA) target recognition is largely dictated by short 'seed' sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA-target interactions to the regulation of biological processes in vivo remains challenging. Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA-MRE interactions at any point during development.

  15. Mapping 3-D functional capillary geometry in rat skeletal muscle in vivo

    PubMed Central

    Milkovich, Stephanie; Goldman, Daniel; Ellis, Christopher G.

    2012-01-01

    We have developed a novel mapping software package to reconstruct microvascular networks in three dimensions (3-D) from in vivo video images for use in blood flow and O2 transport modeling. An intravital optical imaging system was used to collect video sequences of blood flow in microvessels at different depths in the tissue. Functional images of vessels were produced from the video sequences and were processed using automated edge tracking software to yield location and geometry data for construction of the 3-D network. The same video sequences were analyzed for hemodynamic and O2 saturation data from individual capillaries in the network. Simple user-driven commands allowed the connection of vessel segments at bifurcations, and semiautomated registration enabled the tracking of vessels across multiple focal planes and fields of view. The reconstructed networks can be rotated and manipulated in 3-D to verify vessel connections and continuity. Hemodynamic and O2 saturation measurements made in vivo can be indexed to corresponding vessels and visualized using colorized maps of the vascular geometry. Vessels in each reconstruction are saved as text-based files that can be easily imported into flow or O2 transport models with complete geometry, hemodynamic, and O2 transport conditions. The results of digital morphometric analysis of seven microvascular networks showed mean capillary diameters and overall capillary density consistent with previous findings using histology and corrosion cast techniques. The described mapping software is a valuable tool for the quantification of in vivo microvascular geometry, hemodynamics, and oxygenation, thus providing rich data sets for experiment-based computational models. PMID:22140042

  16. Rtfc (4931414P19Rik) Regulates in vitro Thyroid Differentiation and in vivo Thyroid Function

    PubMed Central

    Yu, Yang; Liu, Chang; Zhang, Junxia; Zhang, Mimi; Wen, Wei; Ruan, Xianhui; Li, Dapeng; Zhang, Shuang; Gao, Ming; Chen, Lingyi

    2017-01-01

    Thyroid is a one of the most important endocrine organs. Understanding the molecular mechanism underlying thyroid development and function, as well as thyroid diseases, is beneficial for the clinical treatment of thyroid diseases and tumors. Through genetic linkage analysis and exome sequencing, we previously identified an uncharacterized gene C14orf93 (RTFC, mouse homolog: 4931414P19Rik) as a novel susceptibility gene for familial non-medullary thyroid carcinoma, and demonstrated its function in promoting thyroid tumor. However, the role of RTFC in thyroid development and function remains unexplored. In this study, we found that knockout of Rtfc compromises the in vitro thyroid differentiation of mouse embryonic stem cells. In contrast, Rtfc−/− mice are viable and fertile, and the size and the morphology of thyroid are not affected by Rtfc knockout. However, female Rtfc−/− mice, but not male Rtfc−/− mice, display mild hypothyroidism. In summary, our data suggest the roles of Rtfc in in vitro thyroid differentiation of embryonic stem cells, and in vivo thyroid function. PMID:28230092

  17. Rtfc (4931414P19Rik) Regulates in vitro Thyroid Differentiation and in vivo Thyroid Function.

    PubMed

    Yu, Yang; Liu, Chang; Zhang, Junxia; Zhang, Mimi; Wen, Wei; Ruan, Xianhui; Li, Dapeng; Zhang, Shuang; Gao, Ming; Chen, Lingyi

    2017-02-23

    Thyroid is a one of the most important endocrine organs. Understanding the molecular mechanism underlying thyroid development and function, as well as thyroid diseases, is beneficial for the clinical treatment of thyroid diseases and tumors. Through genetic linkage analysis and exome sequencing, we previously identified an uncharacterized gene C14orf93 (RTFC, mouse homolog: 4931414P19Rik) as a novel susceptibility gene for familial non-medullary thyroid carcinoma, and demonstrated its function in promoting thyroid tumor. However, the role of RTFC in thyroid development and function remains unexplored. In this study, we found that knockout of Rtfc compromises the in vitro thyroid differentiation of mouse embryonic stem cells. In contrast, Rtfc(-/-) mice are viable and fertile, and the size and the morphology of thyroid are not affected by Rtfc knockout. However, female Rtfc(-/-) mice, but not male Rtfc(-/-) mice, display mild hypothyroidism. In summary, our data suggest the roles of Rtfc in in vitro thyroid differentiation of embryonic stem cells, and in vivo thyroid function.

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

    Cancer.gov

    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.

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

    SciTech Connect

    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.

  20. Inflammation modulates human HDL composition and function in vivo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed

    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.

  2. 40 CFR 798.5385 - In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Genetic Toxicity § 798.5385 In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis. (a... intralaboratory variation with the in vivo bone morrow metaphase procedure,” Handbook of mutagenicity...

  3. Longitudinal Functional Data Analysis.

    PubMed

    Park, So Young; Staicu, Ana-Maria

    We consider dependent functional data that are correlated because of a longitudinal-based design: each subject is observed at repeated times and at each time a functional observation (curve) is recorded. We propose a novel parsimonious modeling framework for repeatedly observed functional observations that allows to extract low dimensional features. The proposed methodology accounts for the longitudinal design, is designed to study the dynamic behavior of the underlying process, allows prediction of full future trajectory, and is computationally fast. Theoretical properties of this framework are studied and numerical investigations confirm excellent behavior in finite samples. The proposed method is motivated by and applied to a diffusion tensor imaging study of multiple sclerosis.

  4. Specific in vivo knockdown of protein function by intrabodies

    PubMed Central

    Marschall, Andrea LJ; Dübel, Stefan; Böldicke, Thomas

    2015-01-01

    Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e., their antigens). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, miRNA and small molecule inhibitors, by-passing various limitations and disadvantages of these methods. The advantages of intrabodies include very high specificity for the target, the possibility to knock down several protein isoforms by one intrabody and targeting of specific splice variants or even post-translational modifications. Different types of intrabodies must be designed to target proteins at different locations, typically either in the cytoplasm, in the nucleus or in the endoplasmic reticulum (ER). Most straightforward is the use of intrabodies retained in the ER (ER intrabodies) to knock down the function of proteins passing the ER, which disturbs the function of members of the membrane or plasma proteomes. More effort is needed to functionally knock down cytoplasmic or nuclear proteins because in this case antibodies need to provide an inhibitory effect and must be able to fold in the reducing milieu of the cytoplasm. In this review, we present a broad overview of intrabody technology, as well as applications both of ER and cytoplasmic intrabodies, which have yielded valuable insights in the biology of many targets relevant for drug development, including α-synuclein, TAU, BCR-ABL, ErbB-2, EGFR, HIV gp120, CCR5, IL-2, IL-6, β-amyloid protein and p75NTR. Strategies for the generation of intrabodies and various designs of their applications are also reviewed. PMID:26252565

  5. Development of functional in vivo imaging of cerebral lenticulostriate artery using novel synchrotron radiation angiography

    NASA Astrophysics Data System (ADS)

    Lin, Xiaojie; Miao, Peng; Mu, Zhihao; Jiang, Zhen; Lu, Yifan; Guan, Yongjing; Chen, Xiaoyan; Xiao, Tiqiao; Wang, Yongting; Yang, Guo-Yuan

    2015-02-01

    The lenticulostriate artery plays a vital role in the onset and development of cerebral ischemia. However, current imaging techniques cannot assess the in vivo functioning of small arteries such as the lenticulostriate artery in the brain of rats. Here, we report a novel method to achieve a high resolution multi-functional imaging of the cerebrovascular system using synchrotron radiation angiography, which is based on spatio-temporal analysis of contrast density in the arterial cross section. This method provides a unique tool for studying the sub-cortical vascular elasticity after cerebral ischemia in rats. Using this technique, we demonstrated that the vascular elasticity of the lenticulostriate artery decreased from day 1 to day 7 after transient middle cerebral artery occlusion in rats and recovered from day 7 to day 28 compared to the controls (p < 0.001), which paralleled with brain edema formation and inversely correlated with blood flow velocity (p < 0.05). Our results demonstrated that the change of vascular elasticity was related to the levels of brain edema and the velocity of focal blood flow, suggesting that reducing brain edema is important for the improvement of the function of the lenticulostriate artery in the ischemic brain.

  6. Function Analysis and Decomposistion using Function Analysis Systems Technique

    SciTech Connect

    J. R. Wixson

    1999-06-01

    The "Father of Value Analysis", Lawrence D. Miles, was a design engineer for General Electric in Schenectady, New York. Miles developed the concept of function analysis to address difficulties in satisfying the requirements to fill shortages of high demand manufactured parts and electrical components during World War II. His concept of function analysis was further developed in the 1960s by Charles W. Bytheway, a design engineer at Sperry Univac in Salt Lake City, Utah. Charles Bytheway extended Mile's function analysis concepts and introduced the methodology called Function Analysis Systems Techniques (FAST) to the Society of American Value Engineers (SAVE) at their International Convention in 1965 (Bytheway 1965). FAST uses intuitive logic to decompose a high level, or objective function into secondary and lower level functions that are displayed in a logic diagram called a FAST model. Other techniques can then be applied to allocate functions to components, individuals, processes, or other entities that accomplish the functions. FAST is best applied in a team setting and proves to be an effective methodology for functional decomposition, allocation, and alternative development.

  7. Function Analysis and Decomposistion using Function Analysis Systems Technique

    SciTech Connect

    Wixson, James Robert

    1999-06-01

    The "Father of Value Analysis", Lawrence D. Miles, was a design engineer for General Electric in Schenectady, New York. Miles developed the concept of function analysis to address difficulties in satisfying the requirements to fill shortages of high demand manufactured parts and electrical components during World War II. His concept of function analysis was further developed in the 1960s by Charles W. Bytheway, a design engineer at Sperry Univac in Salt Lake City, Utah. Charles Bytheway extended Mile's function analysis concepts and introduced the methodology called Function Analysis Systems Technique (FAST) to the Society of American Value Engineers (SAVE) at their International Convention in 1965 (Bytheway 1965). FAST uses intuitive logic to decompose a high level, or objective function into secondary and lower level functions that are displayed in a logic diagram called a FAST model. Other techniques can then be applied to allocate functions to components, individuals, processes, or other entities that accomplish the functions. FAST is best applied in a team setting and proves to be an effective methodology for functional decomposition, allocation, and alternative development.

  8. Functional Group Analysis.

    ERIC Educational Resources Information Center

    Smith, Walter T., Jr.; Patterson, John M.

    1984-01-01

    Literature on analytical methods related to the functional groups of 17 chemical compounds is reviewed. These compounds include acids, acid azides, alcohols, aldehydes, ketones, amino acids, aromatic hydrocarbons, carbodiimides, carbohydrates, ethers, nitro compounds, nitrosamines, organometallic compounds, peroxides, phenols, silicon compounds,…

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

    PubMed Central

    Tao, Can; Zhang, Guangwei; Xiong, Ying; Zhou, Yi

    2015-01-01

    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. PMID:26052270

  10. In vivo Analysis of Choroid Plexus Morphogenesis in Zebrafish

    PubMed Central

    Fong, Steven H.; Ye, Zhang-Rui; Korzh, Vladimir

    2008-01-01

    Background The choroid plexus (ChP), a component of the blood-brain barrier (BBB), produces the cerebrospinal fluid (CSF) and as a result plays a role in (i) protecting and nurturing the brain as well as (ii) in coordinating neuronal migration during neurodevelopment. Until now ChP development was not analyzed in living vertebrates due to technical problems. Methodology/Principal Findings We have analyzed the formation of the fourth ventricle ChP of zebrafish in the GFP-tagged enhancer trap transgenic line SqET33-E20 (Gateways) by a combination of in vivo imaging, histology and mutant analysis. This process includes the formation of the tela choroidea (TC), the recruitment of cells from rhombic lips and, finally, the coalescence of TC resulting in formation of ChP. In Notch-deficient mib mutants the first phase of this process is affected with premature GFP expression, deficient cell recruitment into TC and abnormal patterning of ChP. In Hedgehog-deficient smu mutants the second phase of the ChP morphogenesis lacks cell recruitment and TC cells undergo apoptosis. Conclusions/Significance This study is the first to demonstrate the formation of ChP in vivo revealing a role of Notch and Hedgehog signalling pathways during different developmental phases of this process. PMID:18769618

  11. 40 CFR 798.5385 - In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 33 2012-07-01 2012-07-01 false In vivo mammalian bone marrow... Genetic Toxicity § 798.5385 In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis. (a) Purpose. The in vivo bone marrow cytogenetic test is a mutagenicity test for the detection of...

  12. 40 CFR 798.5385 - In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 32 2014-07-01 2014-07-01 false In vivo mammalian bone marrow... Genetic Toxicity § 798.5385 In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis. (a) Purpose. The in vivo bone marrow cytogenetic test is a mutagenicity test for the detection of...

  13. SAHA enhances synaptic function and plasticity in vitro but has limited brain availability in vivo and does not impact cognition.

    PubMed

    Hanson, Jesse E; La, Hank; Plise, Emile; Chen, Yung-Hsiang; Ding, Xiao; Hanania, Taleen; Sabath, Emily V; Alexandrov, Vadim; Brunner, Dani; Leahy, Emer; Steiner, Pascal; Liu, Lichuan; Scearce-Levie, Kimberly; Zhou, Qiang

    2013-01-01

    Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) used for the treatment of cutaneous T cell lymphoma (CTCL) and under consideration for other indications. In vivo studies suggest reducing HDAC function can enhance synaptic function and memory, raising the possibility that SAHA treatment could have neurological benefits. We first examined the impacts of SAHA on synaptic function in vitro using rat organotypic hippocampal brain slices. Following several days of SAHA treatment, basal excitatory but not inhibitory synaptic function was enhanced. Presynaptic release probability and intrinsic neuronal excitability were unaffected suggesting SAHA treatment selectively enhanced postsynaptic excitatory function. In addition, long-term potentiation (LTP) of excitatory synapses was augmented, while long-term depression (LTD) was impaired in SAHA treated slices. Despite the in vitro synaptic enhancements, in vivo SAHA treatment did not rescue memory deficits in the Tg2576 mouse model of Alzheimer's disease (AD). Along with the lack of behavioral impact, pharmacokinetic analysis indicated poor brain availability of SAHA. Broader assessment of in vivo SAHA treatment using high-content phenotypic characterization of C57Bl6 mice failed to demonstrate significant behavioral effects of up to 150 mg/kg SAHA following either acute or chronic injections. Potentially explaining the low brain exposure and lack of behavioral impacts, SAHA was found to be a substrate of the blood brain barrier (BBB) efflux transporters Pgp and Bcrp1. Thus while our in vitro data show that HDAC inhibition can enhance excitatory synaptic strength and potentiation, our in vivo data suggests limited brain availability may contribute to the lack of behavioral impact of SAHA following peripheral delivery. These results do not predict CNS effects of SAHA during clinical use and also emphasize the importance of analyzing brain drug levels when interpreting preclinical

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

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed

    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.

  16. Functional Generalized Structured Component Analysis.

    PubMed

    Suk, Hye Won; Hwang, Heungsun

    2016-12-01

    An extension of Generalized Structured Component Analysis (GSCA), called Functional GSCA, is proposed to analyze functional data that are considered to arise from an underlying smooth curve varying over time or other continua. GSCA has been geared for the analysis of multivariate data. Accordingly, it cannot deal with functional data that often involve different measurement occasions across participants and a large number of measurement occasions that exceed the number of participants. Functional GSCA addresses these issues by integrating GSCA with spline basis function expansions that represent infinite-dimensional curves onto a finite-dimensional space. For parameter estimation, functional GSCA minimizes a penalized least squares criterion by using an alternating penalized least squares estimation algorithm. The usefulness of functional GSCA is illustrated with gait data.

  17. Transcriptomic analysis of neurulation and early organogenesis in rat embryos: an in vivo and ex vivo comparison.

    PubMed

    Robinson, Joshua F; Verhoef, Aart; Piersma, Aldert H

    2012-03-01

    Cultured embryos mimic the morphological developmental progression of embryos (in vivo) undergoing neurulation and early organogenesis. Using available genomics technologies, comparative molecular-based assessments between cultured embryos and in vivo models may further clarify commonalities and dissimilarities, which contribute to differences between systems. Therefore, in this study, using a transcriptomic approach, we compared cultured whole rat embryos and embryos in vivo at comparable time points in development (gestational day (GD) 10 + 2-48 h, GD 0 = copulatory plug) to assess for commonalities and differences in gene expression in relation to morphology. We reveal strong parallels in time-dependent expression of genes in terms of magnitude, directionality, and functionality between whole embryo culture (WEC) and in vivo (rat). Genes changing in expression over time resemble previously hypothesized mechanisms underlying early development in mammalian systems. Furthermore, at the gene and functional level, we identify genes, which differ in expression between models, including genes related to development, oxygen transport, and metabolism. In summary, our results support the use of WEC for toxicological studies aimed at representing in vivo development during this time window at the molecular level. Additionally, we indicate genes, which differ in expression between models, providing possible insights for improvement of culture conditions.

  18. In vivo circulation, clearance, and biodistribution of polyglycerol grafted functional red blood cells.

    PubMed

    Chapanian, Rafi; Constantinescu, Iren; Brooks, Donald E; Scott, Mark D; Kizhakkedathu, Jayachandran N

    2012-04-01

    The in vivo circulation of hyperbranched polyglycerol (HPG) grafted red blood cells (RBCs) was investigated in mice. The number of HPG molecules grafted per RBC was measured using tritium labeled HPGs ((3)H-HPG) of different molecular weights; the values ranged from 1 × 10(5) to 2 × 10(6) molecules per RBC. HPG-grafted RBCs were characterized in vitro by measuring the electrophoretic mobility, complement mediated lysis, and osmotic fragility. Our results show that RBCs grafted with 1.5 × 10(5) HPG molecules per RBC having molecular weights 20 and 60 kDa have similar characteristics as that of control RBCs. The in vivo circulation of HPG-grafted RBCs was measured by a tail vain injection of (3)H-HPG60K-RBC in mice. The radioactivity of isolated RBCs, whole blood, plasma, different organs, urine and feces was evaluated at different time intervals. The portion of (3)H-HPG60K-RBC that survived the first day in mice (52%) remained in circulation for 50 days. Minimal accumulation radioactivity in organs other than liver and spleen was observed suggesting the normal clearance mechanism of modified RBCs. Animals gained normal weights and no abnormalities observed in necropsy analysis. The stability of the ester-amide linker between the RBC and HPG was evaluated by comparing the clearance rate of (3)H-HPG60K-RBC and PKH-26 lipid fluorescent membrane marker labeled HPG60K-RBCs. HPG modified RBCs combine the many advantages of a dendritic polymer and RBCs, and hold great promise in systemic drug delivery and other applications of functional RBC.

  19. Kinetic analysis of pre-ribosome structure in vivo

    PubMed Central

    Swiatkowska, Agata; Wlotzka, Wiebke; Tuck, Alex; Barrass, J. David; Beggs, Jean D.; Tollervey, David

    2012-01-01

    Pre-ribosomal particles undergo numerous structural changes during maturation, but their high complexity and short lifetimes make these changes very difficult to follow in vivo. In consequence, pre-ribosome structure and composition have largely been inferred from purified particles and analyzed in vitro. Here we describe techniques for kinetic analyses of the changes in pre-ribosome structure in living cells of Saccharomyces cerevisiae. To allow this, in vivo structure probing by DMS modification was combined with affinity purification of newly synthesized 20S pre-rRNA over a time course of metabolic labeling with 4-thiouracil. To demonstrate that this approach is generally applicable, we initially analyzed the accessibility of the region surrounding cleavage site D site at the 3′ end of the mature 18S rRNA region of the pre-rRNA. This revealed a remarkably flexible structure throughout 40S subunit biogenesis, with little stable RNA–protein interaction apparent. Analysis of folding in the region of the 18S central pseudoknot was consistent with previous data showing U3 snoRNA–18S rRNA interactions. Dynamic changes in the structure of the hinge between helix 28 (H28) and H44 of pre-18S rRNA were consistent with recently reported interactions with the 3′ guide region of U3 snoRNA. Finally, analysis of the H18 region indicates that the RNA structure matures early, but additional protection appears subsequently, presumably reflecting protein binding. The structural analyses described here were performed on total, affinity-purified, newly synthesized RNA, so many classes of RNA and RNA–protein complex are potentially amenable to this approach. PMID:23093724

  20. Circumferentially aligned fibers guided functional neoartery regeneration in vivo.

    PubMed

    Zhu, Meifeng; Wang, Zhihong; Zhang, Jiamin; Wang, Lina; Yang, Xiaohu; Chen, Jingrui; Fan, Guanwei; Ji, Shenglu; Xing, Cheng; Wang, Kai; Zhao, Qiang; Zhu, Yan; Kong, Deling; Wang, Lianyong

    2015-08-01

    An ideal vascular graft should have the ability to guide the regeneration of neovessels with structure and function similar to those of the native blood vessels. Regeneration of vascular smooth muscle cells (VSMCs) with circumferential orientation within the grafts is crucial for functional vascular reconstruction in vivo. To date, designing and fabricating a vascular graft with well-defined geometric cues to facilitate simultaneously VSMCs infiltration and their circumferential alignment remains a great challenge and scarcely reported in vivo. Thus, we have designed a bi-layered vascular graft, of which the internal layer is composed of circumferentially aligned microfibers prepared by wet-spinning and an external layer composed of random nanofibers prepared by electrospinning. While the internal circumferentially aligned microfibers provide topographic guidance for in vivo regeneration of circumferentially aligned VSMCs, the external random nanofibers can offer enhanced mechanical property and prevent bleeding during and after graft implantation. VSMCs infiltration and alignment within the scaffold was then evaluated in vitro and in vivo. Our results demonstrated that the circumferentially oriented VSMCs and longitudinally aligned ECs were successfully regenerated in vivo after the bi-layered vascular grafts were implanted in rat abdominal aorta. No formation of thrombosis or intimal hyperplasia was observed up to 3 month post implantation. Further, the regenerated neoartery exhibited contraction and relaxation property in response to vasoactive agents. This new strategy may bring cell-free small diameter vascular grafts closer to clinical application.

  1. Recent developments in the understanding of astrocyte function in the cerebellum in vivo.

    PubMed

    Hoogland, Tycho M; Kuhn, Bernd

    2010-09-01

    Several studies have contributed to our understanding of astrocytes, especially Bergmann glia, in the cerebellum; but, until recently, none has looked at their function in vivo. Multicell bolus loading of fluorescent calcium indicators in combination with the astrocytic marker SR101 has allowed imaging of up to hundreds of astrocytes at once in the intact cerebellum. In addition, the selective targeting of astrocytes with fluorescent calcium indicator proteins has enabled the study of their function in vivo without the confounding effects of other neuropil signals and with a resolution that surpasses multicell bolus loading and SR101 staining. The two astrocyte types of the cerebellar cortex, Bergmann glia, and velate protoplasmic astrocytes display a diverse signaling repertoire in vivo, which ranges from localized calcium elevations in subcellular processes to waves, triggered by the release of purines and mediated by purinergic receptors that span multiple processes and can involve tens of astrocytes. During locomotor behavior, even larger numbers of astrocytes display calcium increases that are driven by neuronal activity and correlate with global changes in blood flow. In this review, we give an overview of our current understanding of the function of Bergmann glia and velate protoplasmic astrocytes and the promise of the tools used to study their calcium dynamics and function in vivo.

  2. EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE F344 RAT

    EPA Science Inventory

    EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE PREGNANT F344 RAT.

    S. R. Bielmeier1, A. S. Murr2, D. S. Best2, J. M. Goldman2, and M. G. Narotsky2

    1 Curriculum in Toxicology, Univ. of North Carolina, Chapel Hill, NC, USA
    2 Reproductive T...

  3. EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE PREGNANT F344 RAT

    EPA Science Inventory

    EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE PREGNANT F344 RAT.

    S. R. Bielmeier1, A. S. Murr2, D. S. Best2, J. M. Goldman2, and M. G. Narotsky2

    1 Curriculum in Toxicology, Univ. of North Carolina, Chapel Hill, NC, USA
    2 Reproductive T...

  4. EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE F344 RAT DURING PREGNANCY

    EPA Science Inventory

    Effects of Bromodichloromethane (BDCM) on Ex Vivo Luteal Function In the Pregnant F344 Rat

    Susan R. Bielmeier1, Ashley S. Murr2, Deborah S. Best2, Jerome M. Goldman2, and Michael G. Narotsky2

    1Curriculum in Toxicology, Univ. of North Carolina, Chapel Hill, NC 27599,...

  5. AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia

    PubMed Central

    Ruozi, Giulia; Bortolotti, Francesca; Falcione, Antonella; Dal Ferro, Matteo; Ukovich, Laura; Macedo, Antero; Zentilin, Lorena; Filigheddu, Nicoletta; Cappellari, Gianluca Gortan; Baldini, Giovanna; Zweyer, Marina; Barazzoni, Rocco; Graziani, Andrea; Zacchigna, Serena; Giacca, Mauro

    2015-01-01

    Functional screening of expression libraries in vivo would offer the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we describe a procedure for the functional selection of tissue-protective factors based on the in vivo delivery of arrayed cDNA libraries from the mouse secretome using adeno-associated virus (AAV) vectors. Application of this technique, which we call FunSel, in the context of acute ischaemia, revealed that the peptide ghrelin protects skeletal muscle and heart from ischaemic damage. When delivered to the heart using an AAV9 vector, ghrelin markedly reduces infarct size and preserves cardiac function over time. This protective activity associates with the capacity of ghrelin to sustain autophagy and remove dysfunctional mitochondria after myocardial infarction. Our findings describe an innovative tool to identify biological therapeutics and reveal a novel role of ghrelin as an inducer of myoprotective autophagy. PMID:26066847

  6. Functional dynamics of hippocampal glutamate during associative learning assessed with in vivo (1)H functional magnetic resonance spectroscopy.

    PubMed

    Stanley, Jeffrey A; Burgess, Ashley; Khatib, Dalal; Ramaseshan, Karthik; Arshad, Muzamil; Wu, Helen; Diwadkar, Vaibhav A

    2017-03-29

    fMRI has provided vibrant characterization of regional and network responses associated with associative learning and memory; however, their relationship to functional neurochemistry is unclear. Here, we introduce a novel application of in vivo proton functional magnetic resonance spectroscopy ((1)H fMRS) to investigate the dynamics of hippocampal glutamate during paired-associated learning and memory in healthy young adults. We show that the temporal dynamics of glutamate differed significantly during processes of memory consolidation and retrieval. Moreover, learning proficiency was predictive of the temporal dynamics of glutamate such that fast learners were characterized by a significant increase in glutamate levels early in learning, whereas this increase was only observed later in slow learners. The observed functional dynamics of glutamate provides a novel in vivo marker of brain function. Previously demonstrated N-methyl-D-aspartate (NMDA) receptor mediated synaptic plasticity during associative memory formation may be expressed in glutamate dynamics, which the novel application of (1)H MRS is sensitive to. The novel application of (1)H fMRS can provide highly innovative vistas for characterizing brain function in vivo, with significant implications for studying glutamatergic neurotransmission in health and disorders such as schizophrenia.

  7. In Vivo Electrochemical Analysis of a PEDOT/MWCNT Neural Electrode Coating

    PubMed Central

    Alba, Nicolas A.; Du, Zhanhong J.; Catt, Kasey A.; Kozai, Takashi D. Y.; Cui, X. Tracy

    2015-01-01

    Neural electrodes hold tremendous potential for improving understanding of brain function and restoring lost neurological functions. Multi-walled carbon nanotube (MWCNT) and dexamethasone (Dex)-doped poly(3,4-ethylenedioxythiophene) (PEDOT) coatings have shown promise to improve chronic neural electrode performance. Here, we employ electrochemical techniques to characterize the coating in vivo. Coated and uncoated electrode arrays were implanted into rat visual cortex and subjected to daily cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) for 11 days. Coated electrodes experienced a significant decrease in 1 kHz impedance within the first two days of implantation followed by an increase between days 4 and 7. Equivalent circuit analysis showed that the impedance increase is the result of surface capacitance reduction, likely due to protein and cellular processes encapsulating the porous coating. Coating’s charge storage capacity remained consistently higher than uncoated electrodes, demonstrating its in vivo electrochemical stability. To decouple the PEDOT/MWCNT material property changes from the tissue response, in vitro characterization was conducted by soaking the coated electrodes in PBS for 11 days. Some coated electrodes exhibited steady impedance while others exhibiting large increases associated with large decreases in charge storage capacity suggesting delamination in PBS. This was not observed in vivo, as scanning electron microscopy of explants verified the integrity of the coating with no sign of delamination or cracking. Despite the impedance increase, coated electrodes successfully recorded neural activity throughout the implantation period. PMID:26473938

  8. Quantifying long-term microelectrode array functionality using chronic in vivo impedance testing

    NASA Astrophysics Data System (ADS)

    Prasad, Abhishek; Sanchez, Justin C.

    2012-04-01

    Long-term acquisition of high-quality neural recordings is a cornerstone of neuroprosthetic system design. Mitigating the experimental variability of chronically implanted arrays has been a formidable task because the sensor recording sites can be influenced by biotic and abiotic responses. Several studies have implicated changes in electrical interface impedance as a preliminary marker to infer electrode viability. Microelectrode impedance plays an important role in the monitoring of low amplitude and high-resolution extracellular neural signals. In this work, we seek to quantify long-term microelectrode array functionality and derive an impedance-based predictor for electrode functionality that correlates the recording site electrical properties with the functional neuronal recordings in vivo. High temporal resolution metrics of this type would allow one to assess, predict, and improve electrode performance in the future. In a large cohort of animals, we performed daily impedance measurements and neural signal recordings over long periods (up to 21 weeks) of time in rats using tungsten microwire arrays implanted into the somatosensory cortex. This study revealed that there was a time-varying trend in the modulation of impedance that was related to electrode performance. Single units were best detected from electrodes at time points when the electrode entered into the 40-150 KΩ impedance range. This impedance trend was modeled across the full cohort of animals to predict future electrode performance. The model was tested on data from all animals and was able to provide predictions of electrode performance chronically. Insight from this study can be combined with knowledge of electrode materials and histological analysis to provide a more comprehensive predictive model of electrode failure in the future.

  9. Birefringence Microscopy Platform for Assessing Airway Smooth Muscle Structure and Function in vivo

    PubMed Central

    Adams, David C.; Hariri, Lida P.; Miller, Alyssa J.; Wang, Yan; Cho, Josalyn L.; Villiger, Martin; Holz, Jasmin A.; Szabari, Margit V.; Hamilos, Daniel L.; Harris, R. Scott; Griffith, Jason W.; Bouma, Brett E.; Luster, Andrew D.; Medoff, Benjamin D.; Suter, Melissa J.

    2017-01-01

    The inability to visualize airway smooth muscle (ASM) cells in vivo is a major obstacle in understanding their role in normal physiology and diseases. At present, there is no imaging modality available to assess ASM in vivo. Confocal endomicroscopy lacks the penetration depth and field of view, and conventional optical coherence tomography (OCT) does not have sufficient contrast to differentiate ASM from surrounding tissues. We have developed a birefringence microscopy platform which leverages the micro-organization of tissue to add further dimension to traditional OCT. We have utilized this technology to validate ASM measurements in ex vivo swine and canine studies, visualize and characterize volumetric representations of ASM in vivo, and to quantify and predict ASM contractile force as a function of optical retardation. We provide in vivo images and volumetric assessments of ASM in living humans and document structural disease variations in subjects with mild asthma. The opportunity to link inflammatory responses to ASM responses, and to link ASM responses to clinical responses and outcomes could lead to an increased understanding of diseases of the airway and ultimately to improved patient outcomes. PMID:27708064

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

    NASA Astrophysics Data System (ADS)

    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.

  11. In Vivo Quantification Reveals Extensive Natural Variation in Mitochondrial Form and Function in Caenorhabditis briggsae

    PubMed Central

    Hicks, Kiley A.; Howe, Dana K.; Leung, Aubrey; Denver, Dee R.; Estes, Suzanne

    2012-01-01

    We have analyzed natural variation in mitochondrial form and function among a set of Caenorhabditis briggsae isolates known to harbor mitochondrial DNA structural variation in the form of a heteroplasmic nad5 gene deletion (nad5Δ) that correlates negatively with organismal fitness. We performed in vivo quantification of 24 mitochondrial phenotypes including reactive oxygen species level, membrane potential, and aspects of organelle morphology, and observed significant among-isolate variation in 18 traits. Although several mitochondrial phenotypes were non-linearly associated with nad5Δ levels, most of the among-isolate phenotypic variation could be accounted for by phylogeographic clade membership. In particular, isolate-specific mitochondrial membrane potential was an excellent predictor of clade membership. We interpret this result in light of recent evidence for local adaptation to temperature in C. briggsae. Analysis of mitochondrial-nuclear hybrid strains provided support for both mtDNA and nuclear genetic variation as drivers of natural mitochondrial phenotype variation. This study demonstrates that multicellular eukaryotic species are capable of extensive natural variation in organellar phenotypes and highlights the potential of integrating evolutionary and cell biology perspectives. PMID:22952781

  12. Membrane immunoglobulin expressed by retroviral vector gene transfer mimics partial function of the B-cell receptor in vivo.

    PubMed

    Lu, Jing; Chen, Feng; Xu, Zhen; Zhang, Lingling; Xu, Peng; Liu, Depei; Liang, Chihchuan

    2016-01-01

    Activation of B-cells is initiated by the ligation of B-cell receptors by its cognate antigen, inducing a series of signal cascades. Understanding the molecular mechanisms of these important events is a crucial goal for immunologists. Chimeric B cell receptors provide a powerful tool for analysis of B-cell signal function. However, this method can only be used in tool cells, but cannot be used for in vivo study. Here, we constructed a retroviral vector to encode both heavy chains and light chains of a membrane immunoglobulin, and expressed them in primary B-cells using retroviral gene transfer. Our results demonstrate that the membrane immunoglobulin expressed by retroviral vectors transfer can initiate B-cell receptor-mediated signaling, resulting in the phosphorylation of Syk and Erk1/2 proteins. The results showed that B-cells expressing membrane immunoglobulin can make proliferative responses to cognate antigen both in vitro and in vivo. Therefore, we provide a methodology for rapidly analyzing the downstream signals of B-cell receptors both in vitro and in vivo, which could expedite the identification of proteins involved in B-cell function.

  13. In Vivo Enhancer Analysis Chromosome 16 Conserved NoncodingSequences

    SciTech Connect

    Pennacchio, Len A.; Ahituv, Nadav; Moses, Alan M.; Nobrega,Marcelo; Prabhakar, Shyam; Shoukry, Malak; Minovitsky, Simon; Visel,Axel; Dubchak, Inna; Holt, Amy; Lewis, Keith D.; Plajzer-Frick, Ingrid; Akiyama, Jennifer; De Val, Sarah; Afzal, Veena; Black, Brian L.; Couronne, Olivier; Eisen, Michael B.; Rubin, Edward M.

    2006-02-01

    The identification of enhancers with predicted specificitiesin vertebrate genomes remains a significant challenge that is hampered bya lack of experimentally validated training sets. In this study, weleveraged extreme evolutionary sequence conservation as a filter toidentify putative gene regulatory elements and characterized the in vivoenhancer activity of human-fish conserved and ultraconserved1 noncodingelements on human chromosome 16 as well as such elements from elsewherein the genome. We initially tested 165 of these extremely conservedsequences in a transgenic mouse enhancer assay and observed that 48percent (79/165) functioned reproducibly as tissue-specific enhancers ofgene expression at embryonic day 11.5. While driving expression in abroad range of anatomical structures in the embryo, the majority of the79 enhancers drove expression in various regions of the developingnervous system. Studying a set of DNA elements that specifically droveforebrain expression, we identified DNA signatures specifically enrichedin these elements and used these parameters to rank all ~;3,400human-fugu conserved noncoding elements in the human genome. The testingof the top predictions in transgenic mice resulted in a three-foldenrichment for sequences with forebrain enhancer activity. These datadramatically expand the catalogue of in vivo-characterized human geneenhancers and illustrate the future utility of such training sets for avariety of iological applications including decoding the regulatoryvocabulary of the human genome.

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

    DOE PAGES

    Yaung, Stephanie J.; Deng, Luxue; Li, Ning; ...

    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. Populationmore » 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. Here, our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.« less

  15. Analysis of in vivo somatic mutations in normal human cells

    SciTech Connect

    Gupta, P.K.; Sahota, A.; Boyadjiev, S.A.

    1994-09-01

    We have used the APRT locus located at 16q24.3 to study the nature of loss of heterozygosity (LOH) in human T lymphocytes in vivo. T lymphocytes were isolated from blood from APRT (+/{minus}) obligated heterozygotes with known germline mutations. The cells were immediatley placed in culture medium containing 100 {mu}M 2,6-diaminopurine (DAP) to select for drug-resistant clones ({minus}/{minus}) already present. These clones were first examined using polymorphic CA microsatellite repeat markers D16S303 and D16S305 that are distal and proximal to APRT, respectively. The retention of heterozygosity of these markers is suggestive of minor changes in the APRT gene, the exact nature of which were determined by DNA sequencing. Nineteen out of 70 DAP-resistant clones from one heterozygote showed APRT sequence changes. The loss of heterozygosity of markers D16S303 and D16S305 in the remaining clones suggests LOH involving multilocus chromosomal events. These clones were then sequentially typed using additional CA repeat markers proximal and distal to APRT. The extent of LOH in these clones was found to vary from <5 cM to almost the entire 16q arm. Preliminary results suggest that there are multiple sites along the chromosome from which LOH proceeds distally in these clones. Cytogenetic analysis of 10 clones suggested mitotic recombination in 9 and deletion in one. Studies are in progress to further characterize the molecular mechanisms of LOH.

  16. In Vivo Brillouin Analysis of the Aging Crystalline Lens

    PubMed Central

    Besner, Sebastien; Scarcelli, Giuliano; Pineda, Roberto; Yun, Seok-Hyun

    2016-01-01

    Purpose To analyze the age dependence of the longitudinal modulus of the crystalline lens in vivo using Brillouin scattering data in healthy subjects. Methods Brillouin scans were performed along the crystalline lens in 56 eyes from 30 healthy subjects aged from 19 to 63 years. Longitudinal elastic modulus was acquired along the sagittal axis of the lens with a transverse and axial resolution of 4 and 60 μm, respectively. The relative lens stiffness was computed, and correlations with age were analyzed. Results Brillouin axial profiles revealed nonuniform longitudinal modulus within the lens, increasing from a softer periphery toward a stiffer central plateau at all ages. The longitudinal modulus at the central plateau showed no age dependence in a range of 19 to 45 years and a slight decrease with age from 45 to 63 years. A significant intersubject variability was observed in an age-matched analysis. Importantly, the extent of the central stiff plateau region increased steadily over age from 19 to 63 years. The slope of change in Brillouin modulus in the peripheral regions were nearly age-invariant. Conclusions The adult human lens showed no measurable age-related increase in the peak longitudinal modulus. The expansion of the stiff central region of the lens is likely to be the major contributing factor to age-related lens stiffening. Brillouin microscopy may be useful in characterizing the crystalline lens for the optimization of surgical or pharmacological treatments aimed at restoring accommodative power. PMID:27699407

  17. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9

    PubMed Central

    Swiech, Lukasz; Heidenreich, Matthias; Banerjee, Abhishek; Habib, Naomi; Li, Yinqing; Trombetta, John; Sur, Mriganka; Zhang, Feng

    2015-01-01

    Probing gene function in the mammalian brain can be greatly assisted with methods to manipulate the genome of neurons in vivo. The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9)1 can be used to edit single or multiple genes in replicating eukaryotic cells, resulting in frame-shifting insertion/deletion (indel) mutations and subsequent protein depletion. Here, we delivered SpCas9 and guide RNAs using adeno-associated viral (AAV) vectors to target single (Mecp2) as well as multiple genes (Dnmt1, Dnmt3a and Dnmt3b) in the adult mouse brain in vivo. We characterized the effects of genome modifications in postmitotic neurons using biochemical, genetic, electrophysiological and behavioral readouts. Our results demonstrate that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain. PMID:25326897

  18. Mouse Models for Studies of In Vivo Functions of HIV-1 Nef.

    PubMed

    Zou, Wei; Zhang, Lunli

    2016-01-01

    In vitro studies have demonstrated that HIV-1 Nef has several important activities, promoting viral replication and pathogenesis. These activities include downregulation of cell surface molecules CD4 and major histocompatibility complex class I, enhancement of viral infectivity, activation of p21-activated kinase 2, and inhibition of immunoglobulin class switching. But how important each in vitro activity is to in vivo Nef function remains elusive. To address this question, several small animal models have been developed in the past two decades, such as Nef transgenic mice, SCID-hu mice, and humanized mice. Each of those models has its own pros and cons. Easy access and relative inexpensiveness have made small animal models the favorite models for HIV research. This review will be focused on the recent progress in the understanding of the in vivo functions of HIV-1 Nef obtained from studies using these small animal models.

  19. In vivo generation of a mature and functional artificial skeletal muscle.

    PubMed

    Fuoco, Claudia; Rizzi, Roberto; Biondo, Antonella; Longa, Emanuela; Mascaro, Anna; Shapira-Schweitzer, Keren; Kossovar, Olga; Benedetti, Sara; Salvatori, Maria L; Santoleri, Sabrina; Testa, Stefano; Bernardini, Sergio; Bottinelli, Roberto; Bearzi, Claudia; Cannata, Stefano M; Seliktar, Dror; Cossu, Giulio; Gargioli, Cesare

    2015-04-01

    Extensive loss of skeletal muscle tissue results in mutilations and severe loss of function. In vitro-generated artificial muscles undergo necrosis when transplanted in vivo before host angiogenesis may provide oxygen for fibre survival. Here, we report a novel strategy based upon the use of mouse or human mesoangioblasts encapsulated inside PEG-fibrinogen hydrogel. Once engineered to express placental-derived growth factor, mesoangioblasts attract host vessels and nerves, contributing to in vivo survival and maturation of newly formed myofibres. When the graft was implanted underneath the skin on the surface of the tibialis anterior, mature and aligned myofibres formed within several weeks as a complete and functional extra muscle. Moreover, replacing the ablated tibialis anterior with PEG-fibrinogen-embedded mesoangioblasts also resulted in an artificial muscle very similar to a normal tibialis anterior. This strategy opens the possibility for patient-specific muscle creation for a large number of pathological conditions involving muscle tissue wasting.

  20. Construction of a functional silk-based biomaterial complex with immortalized chondrocytes in vivo.

    PubMed

    Ni, Yusu; Jiang, Yi; Wen, Jianchuan; Shao, Zhenzhong; Chen, Xin; Sun, Shan; Yu, Huiqian; Li, Wen

    2014-04-01

    To explore the feasibility of constructing a functional biomaterial complex with regenerated silk fibroin membrane and immortalized chondrocytes in vivo. Rat auricular chondrocytes (RACs) were transfected with the lentivirus vector pGC-FU-hTERT-3FLAG or pGC-FU-GFP-3FLAG, encoding the human telomerase reverse transcriptase (hTERT) or GFP gene. The effects of regenerated silk fibroin film on the adhesion, growth of immortalized chondrocytes and expression of collagen II in vitro were analyzed with immunofluorescent histochemistry. Immortalized RACs were transformed. Induction by nutrient medium promoted higher expression levels of collagen II in transformed chondrocytes. The regenerated silk fibroin film was not cytotoxic to immortalized chondrocytes and had no adverse influence on their adhesion. Collagen II expression was good in the immortalized chondrocytes in vivo. The construction of a silk-based biomaterial complex with immortalized chondrocytes may provide a feasible kind of functional biomaterial for the repair of cartilage defects in clinical applications.

  1. Generation of Functional Kidney Organoids In Vivo Starting from a Single-Cell Suspension.

    PubMed

    Benedetti, Valentina; Brizi, Valerio; Xinaris, Christodoulos

    2016-08-19

    Novel methods in developmental biology and stem cell research have made it possible to generate complex kidney tissues in vitro that resemble whole organs and are termed organoids. In this chapter we describe a technique using suspensions of fully dissociated mouse kidney cells to yield organoids that can become vascularized in vivo and mature and display physiological functions. This system can be used to produce fine-grained human-mouse chimeric organoids in which the renal differentiation potential of human cells can be assessed. It can also be an excellent method for growing chimeric organoids in vivo using human stem cells, which can differentiate into specialized kidney cells and exert nephron-specific functions. We provide detailed methods, a brief discussion of critical points, and describe some successfully implemented examples of the system.

  2. High throughput in vivo functional validation of candidate congenital heart disease genes in Drosophila.

    PubMed

    Zhu, Jun-Yi; Fu, Yulong; Nettleton, Margaret; Richman, Adam; Han, Zhe

    2017-01-20

    Genomic sequencing has implicated large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system is needed to validate gene associations with pathology. We developed a Drosophila-based functional system to screen candidate disease genes identified from Congenital Heart Disease (CHD) patients. 134 genes were tested in the Drosophila heart using RNAi-based gene silencing. Quantitative analyses of multiple cardiac phenotypes demonstrated essential structural, functional, and developmental roles for more than 70 genes, including a subgroup encoding histone H3K4 modifying proteins. We also demonstrated the use of Drosophila to evaluate cardiac phenotypes resulting from specific, patient-derived alleles of candidate disease genes. We describe the first high throughput in vivo validation system to screen candidate disease genes identified from patients. This approach has the potential to facilitate development of precision medicine approaches for CHD and other diseases associated with genetic factors.

  3. In vivo suppressive function of myeloid-derived suppressor cells is limited to the inflammatory site

    PubMed Central

    Haverkamp, Jessica M.; Crist, Scott A.; Elzey, Bennett D.; Cimen, Cansu; Ratliff, Timothy L.

    2011-01-01

    Current thinking suggests that despite the heterogeneity of myeloid-derived suppressor cells (MDSC), all Gr-1+CD11b+ cells can become suppressive when exposed to inflammatory stimuli. In vitro evaluation shows MDSC from multiple tissue sites have suppressive activity, and in vivo inhibition of MDSC function enhances T cell responses. However, the relative capacity of MDSC present at localized inflammatory sites or in peripheral tissues to suppress T cell responses in vivo has not been directly evaluated. We now demonstrate that during a tissue specific inflammatory response, MDSC inhibition of CD8 T cell proliferation and IFN-γ production is restricted to the inflammatory site. Using a prostate specific inflammatory model and a heterotopic prostate tumor model, we show that MDSC from inflammatory sites or from tumor tissue possess immediate capacity to inhibit T cell function, whereas those isolated from peripheral tissues (spleens and liver) are not suppressive without activation of iNOS by exposure to IFN-γ. These data show MDSC are important regulators of immune responses in the prostate during acute inflammation and the chronic inflammatory setting of tumor growth and that regulation of T cell function by MDSC during a localized inflammatory response is restricted in vivo to the site of an ongoing immune response. PMID:21287554

  4. Astrocyte lipid metabolism is critical for synapse development and function in vivo.

    PubMed

    van Deijk, Anne-Lieke F; Camargo, Nutabi; Timmerman, Jaap; Heistek, Tim; Brouwers, Jos F; Mogavero, Floriana; Mansvelder, Huibert D; Smit, August B; Verheijen, Mark H G

    2017-04-01

    The brain is considered to be autonomous in lipid synthesis with astrocytes producing lipids far more efficiently than neurons. Accordingly, it is generally assumed that astrocyte-derived lipids are taken up by neurons to support synapse formation and function. Initial confirmation of this assumption has been obtained in cell cultures, but whether astrocyte-derived lipids support synapses in vivo is not known. Here, we address this issue and determined the role of astrocyte lipid metabolism in hippocampal synapse formation and function in vivo. Hippocampal protein expression for the sterol regulatory element-binding protein (SREBP) and its target gene fatty acid synthase (Fasn) was found in astrocytes but not in neurons. Diminishing SREBP activity in astrocytes using mice in which the SREBP cleavage-activating protein (SCAP) was deleted from GFAP-expressing cells resulted in decreased cholesterol and phospholipid secretion by astrocytes. Interestingly, SCAP mutant mice showed more immature synapses, lower presynaptic protein SNAP-25 levels as well as reduced numbers of synaptic vesicles, indicating impaired development of the presynaptic terminal. Accordingly, hippocampal short-term and long-term synaptic plasticity were defective in mutant mice. These findings establish a critical role for astrocyte lipid metabolism in presynaptic terminal development and function in vivo. GLIA 2017;65:670-682.

  5. Direct link between RACK1 function and localization at the ribosome in vivo.

    PubMed

    Coyle, Scott M; Gilbert, Wendy V; Doudna, Jennifer A

    2009-03-01

    The receptor for activated C-kinase (RACK1), a conserved protein implicated in numerous signaling pathways, is a stoichiometric component of eukaryotic ribosomes located on the head of the 40S ribosomal subunit. To test the hypothesis that ribosome association is central to the function of RACK1 in vivo, we determined the 2.1-A crystal structure of RACK1 from Saccharomyces cerevisiae (Asc1p) and used it to design eight mutant versions of RACK1 to assess roles in ribosome binding and in vivo function. Conserved charged amino acids on one side of the beta-propeller structure were found to confer most of the 40S subunit binding affinity, whereas an adjacent conserved and structured loop had little effect on RACK1-ribosome association. Yeast mutations that confer moderate to strong defects in ribosome binding mimic some phenotypes of a RACK1 deletion strain, including increased sensitivity to drugs affecting cell wall biosynthesis and translation elongation. Furthermore, disruption of RACK1's position at the 40S ribosomal subunit results in the failure of the mRNA binding protein Scp160 to associate with actively translating ribosomes. These results provide the first direct evidence that RACK1 functions from the ribosome, implying a physical link between the eukaryotic ribosome and cell signaling pathways in vivo.

  6. In vitro and in vivo analysis and characterization of engineered spinal neural implants (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Shor, Erez; Shoham, Shy; Levenberg, Shulamit

    2016-03-01

    Spinal cord injury is a devastating medical condition. Recent developments in pre-clinical and clinical research have started to yield neural implants inducing functional recovery after spinal cord transection injury. However, the functional performance of the transplants was assessed using histology and behavioral experiments which are unable to study cell dynamics and the therapeutic response. Here, we use neurophotonic tools and optogenetic probes to investigate cellular level morphology and activity characteristics of neural implants over time at the cellular level. These methods were used in-vitro and in-vivo, in a mouse spinal cord injury implant model. Following previous attempts to induce recovery after spinal cord injury, we engineered a pre-vascularized implant to obtain better functional performance. To image network activity of a construct implanted in a mouse spinal cord, we transfected the implant to express GCaMP6 calcium activity indicators and implanted these constructs under a spinal cord chamber enabling 2-photon chronic in vivo neural activity imaging. Activity and morphology analysis image processing software was developed to automatically quantify the behavior of the neural and vascular networks. Our experimental results and analyses demonstrate that vascularized and non-vascularized constructs exhibit very different morphologic and activity patterns at the cellular level. This work enables further optimization of neural implants and also provides valuable tools for continuous cellular level monitoring and evaluation of transplants designed for various neurodegenerative disease models.

  7. Determining Protease Activity In Vivo by Fluorescence Cross-Correlation Analysis

    PubMed Central

    Kohl, Tobias; Haustein, Elke; Schwille, Petra

    2005-01-01

    To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions. PMID:16055538

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

    PubMed

    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.

  9. Ex vivo generation of a functional and regenerative wound epithelium from axolotl (Ambystoma mexicanum) skin.

    PubMed

    Ferris, Donald R; Satoh, Akira; Mandefro, Berhan; Cummings, Gillian M; Gardiner, David M; Rugg, Elizabeth L

    2010-10-01

    Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs. Regeneration is a stepwise process that requires interactions between keratinocytes, nerves and fibroblasts. The formation of a wound epithelium covering the amputation site is an early and necessary event in the process but the molecular mechanisms that underlie the role of the wound epithelium in regeneration remain unclear. We have developed an ex vivo model that recapitulates many features of in vivo wound healing. The model comprises a circular explant of axolotl (Ambystoma mexicanum) limb skin with a central circular, full thickness wound. Re-epithelialization of the wound area is rapid (typically <11 h) and is dependent on metalloproteinase activity. The ex vivo wound epithelium is viable, responds to neuronal signals and is able to participate in ectopic blastema formation and limb regeneration. This ex vivo model provides a reproducible and tractable system in which to study the cellular and molecular events that underlie wound healing and regeneration.

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

    PubMed

    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.

  11. Structural and Functional Dissection of the Abp1 ADFH Actin-binding Domain Reveals Versatile In Vivo Adapter Functions

    SciTech Connect

    Quintero-Monzon,O.; Rodal, A.; Strokopytov, B.; Almo, S.; Goode, B.

    2005-01-01

    Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1-actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.

  12. Space station functional relationships analysis

    NASA Technical Reports Server (NTRS)

    Tullis, Thomas S.; Bied, Barbra R.

    1988-01-01

    A systems engineering process is developed to assist Space Station designers to understand the underlying operational system of the facility so that it can be physically arranged and configured to support crew productivity. The study analyzes the operational system proposed for the Space Station in terms of mission functions, crew activities, and functional relationships in order to develop a quantitative model for evaluation of interior layouts, configuration, and traffic analysis for any Station configuration. Development of the model involved identification of crew functions, required support equipment, criteria of assessing functional relationships, and tools for analyzing functional relationship matrices, as well as analyses of crew transition frequency, sequential dependencies, support equipment requirements, potential for noise interference, need for privacy, and overall compatability of functions. The model can be used for analyzing crew functions for the Initial Operating Capability of the Station and for detecting relationships among these functions. Note: This process (FRA) was used during Phase B design studies to test optional layouts of the Space Station habitat module. The process is now being automated as a computer model for use in layout testing of the Space Station laboratory modules during Phase C.

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

    NASA Astrophysics Data System (ADS)

    Hu, Rui; Yong, Ken-Tye; Roy, Indrajit; Ding, Hong; Law, Wing-Cheung; Cai, Hongxing; Zhang, Xihe; Vathy, Lisa A.; Bergey, Earl J.; Prasad, Paras N.

    2010-04-01

    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 CdTe1 - xSex/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 αvβ3 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.

  14. Functional optical coherence tomography for high-resolution mapping of cilia beat frequency in the mouse oviduct in vivo

    NASA Astrophysics Data System (ADS)

    Wang, Shang; Burton, Jason C.; Behringer, Richard R.; Larina, Irina V.

    2016-02-01

    Since mouse is a superior model for genetic analysis of human disorders, reproductive studies in mice have significant implications on further understanding of fertility and infertility in humans. Fertilized oocytes are transported through the reproductive tract by motile cilia lining the lumen of the oviduct as well as by oviduct contractions. While the role of cilia is well recognized, ciliary dynamics in the oviduct is not well understood, largely owing to the lack of live imaging approaches. Here, we report in vivo micro-scale mapping of cilia and cilia beat frequency (CBF) in the mouse oviduct using optical coherence tomography (OCT). This functional imaging method is based on spectral analysis of the OCT speckle variations produced by the beat of cilia in the oviduct, which does not require exogenous contrast agents. Animal procedures similar to the ones used for production of transgenic mice are utilized to expose the reproductive organs for imaging in anesthetized females. In this paper, we first present in vivo structural imaging of the mouse oviduct capturing the oocyte and the preimplantation embryo and then show the result of depth-resolved high-resolution CBF mapping in the ampulla of the live mouse. These data indicate that this structural and functional OCT imaging approach can be a useful tool for a variety of live investigations of mammalian reproduction and infertility.

  15. Skeletal muscle oxidative function in vivo and ex vivo in athletes with marked hypertrophy from resistance training.

    PubMed

    Salvadego, Desy; Domenis, Rossana; Lazzer, Stefano; Porcelli, Simone; Rittweger, Jörn; Rizzo, Giovanna; Mavelli, Irene; Simunic, Bostjan; Pisot, Rado; Grassi, Bruno

    2013-06-01

    Oxidative function during exercise was evaluated in 11 young athletes with marked skeletal muscle hypertrophy induced by long-term resistance training (RTA; body mass 102.6 ± 7.3 kg, mean ± SD) and 11 controls (CTRL; body mass 77.8 ± 6.0 kg). Pulmonary O2 uptake (Vo2) and vastus lateralis muscle fractional O2 extraction (by near-infrared spectroscopy) were determined during an incremental cycle ergometer (CE) and one-leg knee-extension (KE) exercise. Mitochondrial respiration was evaluated ex vivo by high-resolution respirometry in permeabilized vastus lateralis fibers obtained by biopsy. Quadriceps femoris muscle cross-sectional area, volume (determined by magnetic resonance imaging), and strength were greater in RTA vs. CTRL (by ∼40%, ∼33%, and ∼20%, respectively). Vo2peak during CE was higher in RTA vs. CTRL (4.05 ± 0.64 vs. 3.56 ± 0.30 l/min); no difference between groups was observed during KE. The O2 cost of CE exercise was not different between groups. When divided per muscle mass (for CE) or quadriceps muscle mass (for KE), Vo2 peak was lower (by 15-20%) in RTA vs. CTRL. Vastus lateralis fractional O2 extraction was lower in RTA vs. CTRL at all work rates, during both CE and KE. RTA had higher ADP-stimulated mitochondrial respiration (56.7 ± 23.7 pmol O2·s(-1)·mg(-1) ww) vs. CTRL (35.7 ± 10.2 pmol O2·s(-1)·mg(-1) ww) and a tighter coupling of oxidative phosphorylation. In RTA, the greater muscle mass and maximal force and the enhanced mitochondrial respiration seem to compensate for the hypertrophy-induced impaired peripheral O2 diffusion. The net results are an enhanced whole body oxidative function at peak exercise and unchanged efficiency and O2 cost at submaximal exercise, despite a much greater body mass.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  18. Evaluation of effector cell fate and function by in vivo bioluminescence imaging.

    PubMed

    Edinger, Matthias; Hoffmann, Petra; Contag, Christopher H; Negrin, Robert S

    2003-10-01

    The effector functions of immune cells have typically been examined using assays that require sampling of tissues or cells to reveal specific aspects of an immune response (e.g., antigen-specificity, cytokine expression or killing of target cells). The outcome of an immune response in vivo, however, is not solely determined by a single effector function of a specific cell population, but is the result of numerous cellular and molecular interactions that occur in the complex environment of intact organ systems. These interactions influence survival, migration, and activation, as well as final effector function of a given population of cells. Efforts to reveal the cellular and molecular basis of biological processes have resulted in a number of technologies that combine molecular biology and imaging sciences that are collectively termed as Molecular Imaging. This emerging field has developed to reveal functional aspects of cells, genes, and proteins in real time in living animals and humans and embraces multiple modalities, including established clinical imaging methods such as magnetic resonance imaging, single photon emission computed tomography, and positron emission tomography, as well as novel methodologies specifically designed for research animals. Here, we highlight one of the newer modalities, in vivo bioluminescence imaging, as a method for evaluating effector T cell proliferation, migration, and function in model systems of malignant and non-malignant diseases.

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

    PubMed Central

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

    2017-01-01

    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. PMID:28186167

  20. Longitudinal assessment of endothelial function in the microvasculature of mice in-vivo.

    PubMed

    Belch, Jill J F; Akbar, Naveed; Alapati, Venkateswara; Petrie, John; Arthur, Simon; Khan, Faisel

    2013-01-01

    Endothelial dysfunction is associated with early development of cardiovascular disease, making longitudinal measurements desirable. We devised a protocol using laser Doppler imaging (LDI) and iontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP) to assess the skin microcirculation longitudinally in mice every 4 weeks for 24 weeks in two groups of C57BL/6 mice, chow versus high-cholesterol diet(known to induce endothelial dysfunction). LDI measurements were compared with vascular function (isometric tension) measured using wire myography in the tail artery in response to ACh and SNP. Microvascular responses to ACh were significantly reduced in cholesterol-fed versus chow-fed mice from week 4 onwards (P<0.005, ANOVA). Pre-treatment with N(G)-nitro-L-arginine methyl-ester-hydrochloride (L-NAME) showed a significant reduction in ACh response compared with vehicle-treated animals (P<0.05) at baseline and at 12 weeks. In cholesterol-fed mice, ACh responses were 226 ± 21 and 180 ± 21 AU (P=0.03) before and after L-NAME, respectively. A reduction in ex-vivo ACh response was detected in the tail artery in cholesterol-fed mice, and a significant correlation found between peak microvascular ACh response and maximum ACh response in the tail artery (r=0.699, P=0.017). No changes were found in SNP responses in the microvasculature or tail artery. Using this protocol, we have shown longitudinal decreases in microvascular endothelial function to cholesterol feeding. L-NAME studies confirm that the reduced vasodilatation to ACh in cholesterol-fed mice was mediated partly through reduced NO bioavailability. Wire myography of tail arteries confirmed that in-vivo measurements of microvascular function reflect ex-vivo vascular function in other beds. Longitudinal assessments of skin microvascular function in mice could provide a useful translatable model for assessing early endothelial dysfunction.

  1. Dynamic contrast-enhanced optical imaging of in vivo organ function

    PubMed Central

    Wang, Tracy; Bouchard, Matthew B.; McCaslin, Addason F. H.; Blaner, William S.; Levenson, Richard M.; Hillman, Elizabeth M. C.

    2012-01-01

    Abstract. Conventional approaches to optical small animal molecular imaging suffer from poor resolution, limited sensitivity, and unreliable quantitation, often reducing their utility in practice. We previously demonstrated that the in vivo dynamics of an injected contrast agent could be exploited to provide high-contrast anatomical registration, owing to the temporal differences in each organ’s response to the circulating fluorophore. This study extends this approach to explore whether dynamic contrast-enhanced optical imaging (DyCE) can allow noninvasive, in vivo assessment of organ function by quantifying the differing cellular uptake or wash-out dynamics of an agent in healthy and damaged organs. Specifically, we used DyCE to visualize and measure the organ-specific uptake dynamics of indocyanine green before and after induction of transient liver damage. DyCE imaging was performed longitudinally over nine days, and blood samples collected at each imaging session were analyzed for alanine aminotransferase (ALT), a liver enzyme assessed clinically as a measure of liver damage. We show that changes in DyCE-derived dynamics of liver and kidney dye uptake caused by liver damage correlate linearly with ALT concentrations, with an r2 value of 0.91. Our results demonstrate that DyCE can provide quantitative, in vivo, longitudinal measures of organ function with inexpensive and simple data acquisition. PMID:23085904

  2. Dynamic contrast-enhanced optical imaging of in vivo organ function

    NASA Astrophysics Data System (ADS)

    Amoozegar, Cyrus B.; Wang, Tracy; Bouchard, Matthew B.; McCaslin, Addason F. H.; Blaner, William S.; Levenson, Richard M.; Hillman, Elizabeth M. C.

    2012-09-01

    Conventional approaches to optical small animal molecular imaging suffer from poor resolution, limited sensitivity, and unreliable quantitation, often reducing their utility in practice. We previously demonstrated that the in vivo dynamics of an injected contrast agent could be exploited to provide high-contrast anatomical registration, owing to the temporal differences in each organ's response to the circulating fluorophore. This study extends this approach to explore whether dynamic contrast-enhanced optical imaging (DyCE) can allow noninvasive, in vivo assessment of organ function by quantifying the differing cellular uptake or wash-out dynamics of an agent in healthy and damaged organs. Specifically, we used DyCE to visualize and measure the organ-specific uptake dynamics of indocyanine green before and after induction of transient liver damage. DyCE imaging was performed longitudinally over nine days, and blood samples collected at each imaging session were analyzed for alanine aminotransferase (ALT), a liver enzyme assessed clinically as a measure of liver damage. We show that changes in DyCE-derived dynamics of liver and kidney dye uptake caused by liver damage correlate linearly with ALT concentrations, with an r2 value of 0.91. Our results demonstrate that DyCE can provide quantitative, in vivo, longitudinal measures of organ function with inexpensive and simple data acquisition.

  3. EVENT PLANNING USING FUNCTION ANALYSIS

    SciTech Connect

    Lori Braase; Jodi Grgich

    2011-06-01

    Event planning is expensive and resource intensive. Function analysis provides a solid foundation for comprehensive event planning (e.g., workshops, conferences, symposiums, or meetings). It has been used at Idaho National Laboratory (INL) to successfully plan events and capture lessons learned, and played a significant role in the development and implementation of the “INL Guide for Hosting an Event.” Using a guide and a functional approach to planning utilizes resources more efficiently and reduces errors that could be distracting or detrimental to an event. This integrated approach to logistics and program planning – with the primary focus on the participant – gives us the edge.

  4. A functional analysis of crying.

    PubMed

    Bowman, Lynn G; Hardesty, Samantha L; Mendres-Smith, Amber E

    2013-01-01

    Crying has yet to be examined systematically in isolation from other problem behavior, such as aggression or tantrums, during functional analyses (Hanley, Iwata, & McCord, 2003). Identification of variables that may maintain crying is especially important for populations who are susceptible to psychiatric interventions (e.g., individuals who have intellectual disabilities and communication deficits). The current study extended functional analysis methodology to crying with an adolescent boy who had been diagnosed with intellectual disabilities. Results suggested that crying was maintained by caregiver attention delivered in a sympathetic manner.

  5. Functional optoacoustic neuro-tomography of calcium fluxes in adult zebrafish brain in vivo.

    PubMed

    Deán-Ben, X Luís; Gottschalk, Sven; Sela, Gali; Shoham, Shy; Razansky, Daniel

    2017-03-01

    Genetically-encoded calcium indicators (GECIs) have revolutionized neuroimaging by enabling mapping of the activity of entire neuronal populations in vivo. Visualization of these powerful activity sensors has to date been limited to depth-restricted microscopic studies due to intense light scattering in the brain. We demonstrate, for the first time, in vivo real-time volumetric optoacoustic monitoring of calcium transients in adult transgenic zebrafish expressing the GCaMP5G calcium indicator. Fast changes in optoacoustic traces associated with GCaMP5G activity were detectable in the presence of other strongly absorbing endogenous chromophores, such as hemoglobin. The new functional optoacoustic neuroimaging method can visualize neural activity at penetration depths and spatio-temporal resolution scales not covered with the existing neuroimaging techniques.

  6. In vivo NMR for ¹³C Metabolic Flux Analysis.

    PubMed

    Roscher, Albrecht; Troufflard, Stéphanie; Taghki, Abdelghani Idrissi

    2014-01-01

    The use of in vivo NMR within the framework of Metabolic Flux Analysis in plants is presented. In vivo NMR allows to visualize the active metabolic network, to determine metabolic and isotopic steady state and to measure metabolic fluxes which are not necessarily accessible by isotopic steady state (stationary) Metabolic Flux Analysis. The kinetic data can be used as input for dynamic (nonstationary) Metabolic Flux Analysis. Both 1D and 2D NMR methods are employed.

  7. Fluorescent function-spacer-lipid construct labelling allows for real-time in vivo imaging of cell migration and behaviour in zebrafish (Danio rerio).

    PubMed

    Lan, Chuan-Ching; Blake, Deborah; Henry, Stephen; Love, Donald R

    2012-07-01

    Real-time in vivo imaging of cell migration and behavior has advanced our understanding of physiological processes in situ, especially in the field of immunology. We carried out the transplantation of a mixed population of blood cells from adult zebrafish (Danio rerio) to 2 day old embryos. The blood cells were treated ex vivo with Function-Spacer-Lipid constructs (FSL) incorporating either fluorescein or Atto488 fluorophores (FSL-FLRO4-I or -II). Excellent labeling efficiency was demonstrated by epifluorescence microscopy and FACScan analysis. Real-time video imaging of the recipient fish showed that the functionality of these cells was retained and not affected by the labeling. The usefulness of FSL-FLRO4-I as a contrast agent in microangiography was explored. Overall, we found both FSL-FLRO4-I and-II promising labeling dyes for real-time in vivo imaging in zebrafish.

  8. In Vivo Imaging of the Photoreceptor Mosaic in Retinal Dystrophies and Correlations with Visual Function

    PubMed Central

    Choi, Stacey S.; Doble, Nathan; Hardy, Joseph L.; Jones, Steven M.; Keltner, John L.; Olivier, Scot S.; Werner, John S.

    2008-01-01

    Purpose To relate in vivo microscopic retinal changes to visual function in patients who have various forms of retinal dystrophy. Methods 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 fields, multifocal electroretinography (mfERG), and contrast sensitivity were measured in all subjects by using stimuli that were coincident with areas imaged. Five patients with different forms of retinal dystrophy and three control subjects were recruited. Cone densities were quantified for all retinal images. Results 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 apparent in patients with retinal dystrophy. There were significant correlations between functional vision losses and the extent to which these irregularities, quantified by cone density, occurred in retinal images. Conclusions AO fundus imaging 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 provide borderline or ambiguous results, as it allows visualization of individual photoreceptors. PMID:16639019

  9. Serotonin2c receptor constitutive activity: in vivo direct and indirect evidence and functional significance.

    PubMed

    Navailles, Sylvia; Lagière, Mélanie; Guthrie, Martin; De Deurwaerdère, Philippe

    2013-06-01

    Serotonin2c (5-HT2c) receptors are widely expressed in the central nervous system where they play a pivotal role in the regulation of neuronal network excitability. Along with this fundamental physiological function, 5-HT2c receptors are thought to be implicated in the pathophysiology of several neuropsychiatric disorders and have become a major pharmacological target for the development of improved treatments of these disorders. In the past decade, many studies have focused on the constitutive activity of 5-HT2c receptors and the therapeutic potential of drugs acting as inverse agonists. Although the constitutive activity of the 5-HT2c receptor has been clearly described in vitro, the transposition of this concept to living animals is often difficult to ascertain. Nevertheless, cumulating evidence has demonstrated the functional relevance of such property in regulating physiological systems in vivo both at the level of the central and peripheral nervous systems. The present review provides an update of the growing number of studies that show, by means of pharmacological tools, the participation of the constitutive activity of 5-HT2c receptors in the control of various biochemical and behavioural functions in vivo and emphasizes the functional organization of this constitutive control together with the phasic and tonic (involving the spontaneous release of 5-HT) modalities of the 5-HT2c receptor in the brain.

  10. Effects of titanium particle size on osteoblast functions in vitro and in vivo.

    PubMed

    Choi, Moon G; Koh, Hae S; Kluess, Daniel; O'Connor, Daniel; Mathur, Anshu; Truskey, George A; Rubin, Janet; Zhou, David X F; Sung, K-L Paul

    2005-03-22

    The formation of titanium (Ti)-wear particles during the lifetime of an implant is believed to be a major component of loosening due to debris-induced changes in bone cell function. Radiographic evidence indicates a loss of fixation at the implant-bone interface, and we believe that the accumulation of Ti particles may act on the bone-remodeling process and impact both long- and short-term implant-fixation strengths. To determine the effects of various sizes of the Ti particles on osteoblast function in vivo, we measured the loss of integration strength around Ti-pin implants inserted into a rat tibia in conjunction with Ti particles from one of four size-groups. Implant integration is mediated primarily by osteoblast adhesion/focal contact pattern, viability, proliferation and differentiation, and osteoclast recruitment at the implant site in vivo. This study demonstrates the significant attenuation of osteoblast function concurrent with increased expression of receptor activator of nuclear factor kappaB ligand (RANKL), a dominant signal for osteoclast recruitment, which is regulated differentially, depending on the size of the Ti particle. Zymography studies have also demonstrated increased activities of matrix metalloproteinases (MMP) 2 and 9 in cells exposed to larger Ti particles. In summary, all particles have adverse effects on osteoblast function, resulting in decreased bone formation and integration, but different mechanisms are elicited by particles of different sizes.

  11. Analysis of the mutations inducedd by conazole fungicides in vivo

    EPA Science Inventory

    The mouse liver tumorigenic conazo1e fungicides triadimefon and propiconazo1e have previously been shown to be in vivo mouse liver mutagens in the Big Blue" transgenic mutation assay when administered in feed at tumorigenic doses, whereas the nontumorigenic conazo1e myc1obutani1 ...

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

    PubMed Central

    Fröhlich, Eleonore

    2015-01-01

    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. PMID:26060398

  13. Functional evaluation of malaria Pfs25 DNA vaccine by in vivo electroporation in olive baboons.

    PubMed

    Kumar, Rajesh; Nyakundi, Ruth; Kariuki, Thomas; Ozwara, Hastings; Nyamongo, Onkoba; Mlambo, Godfree; Ellefsen, Barry; Hannaman, Drew; Kumar, Nirbhay

    2013-06-28

    Plasmodium falciparum Pfs25 antigen, expressed on the surface of zygotes and ookinetes, is one of the leading targets for the development of a malaria transmission-blocking vaccine (TBV). Our laboratory has been evaluating DNA plasmid based Pfs25 vaccine in mice and non-human primates. Previously, we established that in vivo electroporation (EP) delivery is an effective method to improve the immunogenicity of DNA vaccine encoding Pfs25 in mice. In order to optimize the in vivo EP procedure and test for its efficacy in more clinically relevant larger animal models, we employed in vivo EP to evaluate the immune response and protective efficacy of Pfs25 encoding DNA vaccine in nonhuman primates (olive baboons, Papio anubis). The results showed that at a dose of 2.5mg DNA vaccine, antibody responses were significantly enhanced with EP as compared to without EP resulting in effective transmission blocking efficiency. Similar immunogenicity enhancing effect of EP was also observed with lower doses (0.5mg and 1mg) of DNA plasmids. Further, final boosting with a single dose of recombinant Pfs25 protein resulted in dramatically enhanced antibody titers and significantly increased functional transmission blocking efficiency. Our study suggests priming with DNA vaccine via EP along with protein boost regimen as an effective method to elicit potent immunogenicity of malaria DNA vaccines in nonhuman primates and provides the basis for further evaluation in human volunteers.

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

    PubMed

    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.

  15. Rationally engineered Troponin C modulates in vivo cardiac function and performance in health and disease

    PubMed Central

    Shettigar, Vikram; Zhang, Bo; Little, Sean C.; Salhi, Hussam E.; Hansen, Brian J.; Li, Ning; Zhang, Jianchao; Roof, Steve R.; Ho, Hsiang-Ting; Brunello, Lucia; Lerch, Jessica K.; Weisleder, Noah; Fedorov, Vadim V.; Accornero, Federica; Rafael-Fortney, Jill A.; Gyorke, Sandor; Janssen, Paul M. L.; Biesiadecki, Brandon J.; Ziolo, Mark T.; Davis, Jonathan P.

    2016-01-01

    Treatment for heart disease, the leading cause of death in the world, has progressed little for several decades. Here we develop a protein engineering approach to directly tune in vivo cardiac contractility by tailoring the ability of the heart to respond to the Ca2+ signal. Promisingly, our smartly formulated Ca2+-sensitizing TnC (L48Q) enhances heart function without any adverse effects that are commonly observed with positive inotropes. In a myocardial infarction (MI) model of heart failure, expression of TnC L48Q before the MI preserves cardiac function and performance. Moreover, expression of TnC L48Q after the MI therapeutically enhances cardiac function and performance, without compromising survival. We demonstrate engineering TnC can specifically and precisely modulate cardiac contractility that when combined with gene therapy can be employed as a therapeutic strategy for heart disease. PMID:26908229

  16. Simultaneous functional photoacoustic and ultrasonic endoscopy of internal organs in vivo

    PubMed Central

    Yang, Joon-Mo; Favazza, Christopher; Chen, Ruimin; Yao, Junjie; Cai, Xin; Maslov, Konstantin; Zhou, Qifa; Shung, K. Kirk; Wang, Lihong V.

    2013-01-01

    Presently, clinicians routinely apply ultrasound endoscopy in a variety of interventional procedures which provide treatment solutions for diseased organs. Ultrasound endoscopy not only produces high resolution images, it is also safe for clinical use and broadly applicable. However, for soft tissue imaging, its mechanical wave-based image contrast fundamentally limits its ability to provide physiologically-specific functional information. By contrast, photoacoustic endoscopy possesses a unique combination of functional optical contrast and high spatial resolution at clinically-relevant depths, ideal for soft tissue imaging. With these attributes, photoacoustic endoscopy can overcome the current limitations of ultrasound endoscopy. Moreover, the benefits of photoacoustic imaging do not come at the expense of existing ultrasound functions; photoacoustic endoscopy systems are inherently compatible with ultrasound imaging, enabling multi-modality imaging with complementary contrast. Here, we present simultaneous photoacoustic and ultrasonic dual-mode endoscopy and demonstrate its ability to image internal organs in vivo, illustrating its potential clinical application. PMID:22797808

  17. Ganglionic GFAP (+) glial Gq-GPCR signaling enhances heart functions in vivo.

    PubMed

    Xie, Alison Xiaoqiao; Lee, Jakovin J; McCarthy, Ken D

    2017-01-26

    The sympathetic nervous system (SNS) accelerates heart rate, increases cardiac contractility, and constricts resistance vessels. The activity of SNS efferent nerves is generated by a complex neural network containing neurons and glia. Gq G protein-coupled receptor (Gq-GPCR) signaling in glial fibrillary acidic protein-expressing (GFAP(+)) glia in the central nervous system supports neuronal function and regulates neuronal activity. It is unclear how Gq-GPCR signaling in GFAP(+) glia affects the activity of sympathetic neurons or contributes to SNS-regulated cardiovascular functions. In this study, we investigated whether Gq-GPCR activation in GFAP(+) glia modulates the regulatory effect of the SNS on the heart; transgenic mice expressing Gq-coupled DREADD (designer receptors exclusively activated by designer drugs) (hM3Dq) selectively in GFAP(+) glia were used to address this question in vivo. We found that acute Gq-GPCR activation in peripheral GFAP(+) glia significantly accelerated heart rate and increased left ventricle contraction. Pharmacological experiments suggest that the glial-induced cardiac changes were due to Gq-GPCR activation in satellite glial cells within the sympathetic ganglion; this activation led to increased norepinephrine (NE) release and beta-1 adrenergic receptor activation within the heart. Chronic glial Gq-GPCR activation led to hypotension in female Gfap-hM3Dq mice. This study provides direct evidence that Gq-GPCR activation in peripheral GFAP(+) glia regulates cardiovascular functions in vivo.

  18. Copper-induced changes in reproductive functions: in vivo and in vitro effects.

    PubMed

    Roychoudhury, S; Nath, S; Massanyi, P; Stawarz, R; Kacaniova, M; Kolesarova, A

    2016-01-01

    The goal of this study is to summarize the current knowledge on the effects of one of the essential metals, copper (Cu) on the reproductive system. The development of past four decades addressing effects of Cu on reproductive organs is reviewed. The most relevant data obtained from in vivo and in vitro experiments performed on humans and other mammals, including effects of copper nanoparticles (CuNPs) on the reproductive functions are presented. Short term Cu administration has been found to exert deleterious effect on intracellular organelles of rat ovarian cells in vivo. In vitro administration in porcine ovarian granulosa cells releases insulin-like growth factor (IGF-I), steroid hormone progesterone (P(4)), and induces expression of peptides related to proliferation and apoptosis. Adverse effect of Cu on male reproductive functions has been indicated by the decrease in spermatozoa parameters such as concentration, viability and motility. Copper nanoparticles are capable of generating oxidative stress in vitro thereby leading to reproductive toxicity. Toxic effect of CuNPs has been evident more in male mice than in females. Even though further investigations are necessary to arrive at a definitive conclusion, Cu notably influences the reproductive functions by interfering with both male and female reproductive systems and also hampers embryo development in dose-dependent manner.

  19. Ganglionic GFAP+ glial Gq-GPCR signaling enhances heart functions in vivo

    PubMed Central

    Lee, Jakovin J.; McCarthy, Ken D.

    2017-01-01

    The sympathetic nervous system (SNS) accelerates heart rate, increases cardiac contractility, and constricts resistance vessels. The activity of SNS efferent nerves is generated by a complex neural network containing neurons and glia. Gq G protein–coupled receptor (Gq-GPCR) signaling in glial fibrillary acidic protein–expressing (GFAP+) glia in the central nervous system supports neuronal function and regulates neuronal activity. It is unclear how Gq-GPCR signaling in GFAP+ glia affects the activity of sympathetic neurons or contributes to SNS-regulated cardiovascular functions. In this study, we investigated whether Gq-GPCR activation in GFAP+ glia modulates the regulatory effect of the SNS on the heart; transgenic mice expressing Gq-coupled DREADD (designer receptors exclusively activated by designer drugs) (hM3Dq) selectively in GFAP+ glia were used to address this question in vivo. We found that acute Gq-GPCR activation in peripheral GFAP+ glia significantly accelerated heart rate and increased left ventricle contraction. Pharmacological experiments suggest that the glial-induced cardiac changes were due to Gq-GPCR activation in satellite glial cells within the sympathetic ganglion; this activation led to increased norepinephrine (NE) release and beta-1 adrenergic receptor activation within the heart. Chronic glial Gq-GPCR activation led to hypotension in female Gfap-hM3Dq mice. This study provides direct evidence that Gq-GPCR activation in peripheral GFAP+ glia regulates cardiovascular functions in vivo. PMID:28138563

  20. Small interfering RNAs as a tool to assign Rho GTPase exchange-factor function in vivo.

    PubMed Central

    Gampel, Alexandra; Mellor, Harry

    2002-01-01

    Rho GTPases control a complex network of intracellular signalling pathways. Whereas progress has been made in identifying downstream signalling partners for these proteins, the characterization of Rho upstream regulatory guanine-nucleotide exchange factors (GEFs) has been hampered by a lack of suitable research tools. Here we use small interfering RNAs (siRNAs) to examine the cellular regulation of the RhoB GTPase, and show that RhoB is activated downstream of the epidermal-growth-factor receptor through the Vav2 exchange factor. These studies demonstrate that siRNAs are an ideal research tool for the assignment of Rho GEF function in vivo. PMID:12113653

  1. Minimally invasive microendoscopy system for in vivo functional imaging of deep nuclei in the mouse brain

    PubMed Central

    Bocarsly, Miriam E.; Jiang, Wan-chen; Wang, Chen; Dudman, Joshua T.; Ji, Na; Aponte, Yeka

    2015-01-01

    The ability to image neurons anywhere in the mammalian brain is a major goal of optical microscopy. Here we describe a minimally invasive microendoscopy system for studying the morphology and function of neurons at depth. Utilizing a guide cannula with an ultrathin wall, we demonstrated in vivo two-photon fluorescence imaging of deeply buried nuclei such as the striatum (2.5 mm depth), substantia nigra (4.4 mm depth) and lateral hypothalamus (5.0 mm depth) in mouse brain. We reported, for the first time, the observation of neuronal activity with subcellular resolution in the lateral hypothalamus and substantia nigra of head-fixed awake mice. PMID:26601017

  2. Using CRISPR/Cas to study gene function and model disease in vivo

    PubMed Central

    Tschaharganeh, Darjus F.; Lowe, Scott W.; Garippa, Ralph J.; Livshits, Geulah

    2016-01-01

    The recent discovery of the CRISPR/Cas system and repurposing of this technology to edit a variety of different genomes have revolutionized an array of scientific fields, from genetics and translational research, to agriculture and bioproduction. In particular, the prospect of rapid and precise genome editing in laboratory animals by CRISPR/Cas has generated an immense interest in the scientific community. Here we review current in vivo applications of CRISPR/Cas and how this technology can improve our knowledge of gene function and our understanding of biological processes in animal models. PMID:27149548

  3. Validation of a P-Glycoprotein (P-gp) Humanized Mouse Model by Integrating Selective Absolute Quantification of Human MDR1, Mouse Mdr1a and Mdr1b Protein Expressions with In Vivo Functional Analysis for Blood-Brain Barrier Transport

    PubMed Central

    Sadiq, Muhammad Waqas; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Hammarlund-Udenaes, Margareta

    2015-01-01

    It is essential to establish a useful validation method for newly generated humanized mouse models. The novel approach of combining our established species-specific protein quantification method combined with in vivo functional studies is evaluated to validate a humanized mouse model of P-gp/MDR1 efflux transporter. The P-gp substrates digoxin, verapamil and docetaxel were administered to male FVB Mdr1a/1b(+/+) (FVB WT), FVB Mdr1a/1b(-/-) (Mdr1a/1b(-/-)), C57BL/6 Mdr1a/1b(+/+) (C57BL/6 WT) and humanized C57BL (hMDR1) mice. Brain-to-plasma total concentration ratios (Kp) were measured. Quantitative targeted absolute proteomic (QTAP) analysis was used to selectively quantify the protein expression levels of hMDR1, Mdr1a and Mdr1b in the isolated brain capillaries. The protein expressions of other transporters, receptors and claudin-5 were also quantified. The Kp for digoxin, verapamil, and docetaxel were 20, 30 and 4 times higher in the Mdr1a/1b(-/-) mice than in the FVB WT controls, as expected. The Kp for digoxin, verapamil and docetaxel were 2, 16 and 2-times higher in the hMDR1 compared to the C57BL/6 WT mice. The hMDR1 mice had 63- and 9.1-fold lower expressions of the hMDR1 and Mdr1a proteins than the corresponding expression of Mdr1a in C57BL/6 WT mice, respectively. The protein expression levels of other molecules were almost consistent between C57BL/6 WT and hMDR1 mice. The P-gp function at the BBB in the hMDR1 mice was smaller than that in WT mice due to lower protein expression levels of hMDR1 and Mdr1a. The combination of QTAP and in vivo functional analyses was successfully applied to validate the humanized animal model and evaluates its suitability for further studies. PMID:25932627

  4. Validation of a P-Glycoprotein (P-gp) Humanized Mouse Model by Integrating Selective Absolute Quantification of Human MDR1, Mouse Mdr1a and Mdr1b Protein Expressions with In Vivo Functional Analysis for Blood-Brain Barrier Transport.

    PubMed

    Sadiq, Muhammad Waqas; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Hammarlund-Udenaes, Margareta

    2015-01-01

    It is essential to establish a useful validation method for newly generated humanized mouse models. The novel approach of combining our established species-specific protein quantification method combined with in vivo functional studies is evaluated to validate a humanized mouse model of P-gp/MDR1 efflux transporter. The P-gp substrates digoxin, verapamil and docetaxel were administered to male FVB Mdr1a/1b(+/+) (FVB WT), FVB Mdr1a/1b(-/-) (Mdr1a/1b(-/-)), C57BL/6 Mdr1a/1b(+/+) (C57BL/6 WT) and humanized C57BL (hMDR1) mice. Brain-to-plasma total concentration ratios (Kp) were measured. Quantitative targeted absolute proteomic (QTAP) analysis was used to selectively quantify the protein expression levels of hMDR1, Mdr1a and Mdr1b in the isolated brain capillaries. The protein expressions of other transporters, receptors and claudin-5 were also quantified. The Kp for digoxin, verapamil, and docetaxel were 20, 30 and 4 times higher in the Mdr1a/1b(-/-) mice than in the FVB WT controls, as expected. The Kp for digoxin, verapamil and docetaxel were 2, 16 and 2-times higher in the hMDR1 compared to the C57BL/6 WT mice. The hMDR1 mice had 63- and 9.1-fold lower expressions of the hMDR1 and Mdr1a proteins than the corresponding expression of Mdr1a in C57BL/6 WT mice, respectively. The protein expression levels of other molecules were almost consistent between C57BL/6 WT and hMDR1 mice. The P-gp function at the BBB in the hMDR1 mice was smaller than that in WT mice due to lower protein expression levels of hMDR1 and Mdr1a. The combination of QTAP and in vivo functional analyses was successfully applied to validate the humanized animal model and evaluates its suitability for further studies.

  5. Functional analysis of glaucoma data.

    PubMed

    Hosseini-Nasab, Mohammad; Mirzaei K, Zahra

    2014-05-30

    We refer glaucoma to a category of eye disorders often associated with a dangerous buildup of intraocular pressure (IOP), which can damage the eyes' optic nerve that transmits visual information to the brain. Because IOP changes over time, it is a function of time, and it is an advantage that we analyze the phenomenon using functional data analysis. In this paper, we treat the data related to the IOP of 35 patients with right eye glaucoma, collected in Rasul-e-Akram Hospital at Tehran, Iran, over the years 2007–2011. We shall explore the structure of the data in search of the features that describe them, and find the characteristics that give a comprehensible presentation of the structure of the variability in the data.We extract patterns of variation in the data by using a generalization of the smoothed functional principal component analysis to obtain the main factors causing glaucoma and then determine their importance. We also explore the correlation patterns between the IOP of right and left eyes, and then model the left eye IOP of the glaucoma patients at each time on the basis of their right eye IOP in a previous interval of time.We can use the model to predict the values of the former variable by using the latter one in a previous time interval.

  6. Honokiol as a specific collagen receptor glycoprotein VI antagonist on human platelets: Functional ex vivo and in vivo studies

    PubMed Central

    Lee, Tzu-Yin; Chang, Chao-Chien; Lu, Wan-Jung; Yen, Ting-Lin; Lin, Kuan-Hung; Geraldine, Pitchairaj; Li, Jiun-Yi; Sheu, Joen-Rong

    2017-01-01

    Honokiol, derived from Magnolia officinalis, has various pharmacological properties. Platelet activation plays a critical role in cardiovascular diseases. Honokiol has been reported to inhibit collagen-stimulated rabbit platelet aggregation. However, detailed further studies on the characteristics and functional activity of honokiol in platelet activation are relatively lacking. In the present study, honokiol specifically inhibited platelet aggregation and Ca+2 ion mobilization stimulated with collagen or convulxin, an agonist of glycoprotein (GP) VI, but not with aggretin, an agonist of integrin α2β1. Honokiol also attenuated the phosphorylation of Lyn, PLCγ2, PKC, MAPKs, and Akt after convulxin stimulation. Honokiol have no cytotoxicity in zebrafish embryos. Honokiol diminished the binding of anti-GP VI (FITC-JAQ1) mAb to human platelets, and it also reduced the coimmunoprecipitation of GP VI-bound Lyn after convulxin stimulation. The surface plasmon resonance results revealed that honokiol binds directly to GP VI, with a KD of 289 μM. Platelet function analysis revealed that honokiol substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation in mice. In conclusion, honokiol acts as a potent antagonist of collagen GP VI in human platelets, and it has therapeutic potential in the prevention of the pathological thrombosis. PMID:28054640

  7. Applications of nuclear technologies for in-vivo elemental analysis

    SciTech Connect

    Cohn, S.H.; Ellis, K.J.; Vartsky, D.; Wielopolski, L.

    1982-01-01

    Measurement facilities developed, to date, include a unique whole-body-counter, (WBC); a total-body neutron-activation facility (TBNAA); and a partial-body activation facility (PBNAA). A variation of the prompt-gamma neutron-activation technique for measuring total-body nitrogen was developed to study body composition of cancer patients and the effect of nutritional regimens on the composition. These new techniques provide data in numerous clinical studies not previously amenable to investigation. The development and perfection of these techniques provide unique applications of radiation and radioisotopes to the early diagnosis of certain diseases and the evaluation of therapeutic programs. The PBNAA technique has been developed and calibrated for in-vivo measurement of metals. Development has gone forward on prompt-gamma neutron activation for the measurement of cadmium, x-ray fluorescence (XRF) for measurement of iron. Other techniques are being investigated for in-vivo measurement of metals such as silicon and beryllium.

  8. Novel peptides functionally targeting in vivo human lung cancer discovered by in vivo peptide displayed phage screening.

    PubMed

    Lee, Kyoung Jin; Lee, Jae Hee; Chung, Hye Kyung; Choi, Jinhyang; Park, Jaesook; Park, Seok Soon; Ju, Eun Jin; Park, Jin; Shin, Seol Hwa; Park, Hye Ji; Ko, Eun Jung; Suh, Nayoung; Kim, InKi; Hwang, Jung Jin; Song, Si Yeol; Jeong, Seong-Yun; Choi, Eun Kyung

    2015-02-01

    Discovery of the cancer-specific peptidic ligands have been emphasized for active targeting drug delivery system and non-invasive imaging. For the discovery of useful and applicable peptidic ligands, in vivo peptide-displayed phage screening has been performed in this study using a xenograft mouse model as a mimic microenvironment to tumor. To seek human lung cancer-specific peptides, M13 phage library displaying 2.9 × 10(9) random peptides was intravenously injected into mouse model bearing A549-derived xenograft tumor through the tail vein. Then the phages emerged from a course of four rounds of biopanning in the xenograft tumor tissue. Novel peptides were categorized into four groups according to a sequence-homology phylogenicity, and in vivo tumor-targeting capacity of these peptides was validated by whole body imaging with Cy5.5-labeled phages in various cancer types. The result revealed that novel peptides accumulated only in adenocarcinoma lung cancer cell-derived xenograft tissue. For further confirmation of the specific targeting ability, in vitro cell-binding assay and immunohistochemistry in vivo tumor tissue were performed with a selected peptide. The peptide was found to bind intensely to lung cancer cells both in vitro and in vivo, which was efficiently compromised with unlabeled phages in an in vitro competition assay. In conclusion, the peptides specifically targeting human lung cancer were discovered in this study, which is warranted to provide substantive feasibilities for drug delivery and imaging in terms of a novel targeted therapeutics and diagnostics.

  9. Warped functional analysis of variance.

    PubMed

    Gervini, Daniel; Carter, Patrick A

    2014-09-01

    This article presents an Analysis of Variance model for functional data that explicitly incorporates phase variability through a time-warping component, allowing for a unified approach to estimation and inference in presence of amplitude and time variability. The focus is on single-random-factor models but the approach can be easily generalized to more complex ANOVA models. The behavior of the estimators is studied by simulation, and an application to the analysis of growth curves of flour beetles is presented. Although the model assumes a smooth latent process behind the observed trajectories, smootheness of the observed data is not required; the method can be applied to irregular time grids, which are common in longitudinal studies.

  10. Triboelectric Nanogenerator Accelerates Highly Efficient Nonviral Direct Conversion and In Vivo Reprogramming of Fibroblasts to Functional Neuronal Cells.

    PubMed

    Jin, Yoonhee; Seo, Jungmok; Lee, Jung Seung; Shin, Sera; Park, Hyun-Ji; Min, Sungjin; Cheong, Eunji; Lee, Taeyoon; Cho, Seung-Woo

    2016-09-01

    Triboelectric nanogenerators (TENGs) can be an effective cell reprogramming platform for producing functional neuronal cells for therapeutic applications. Triboelectric stimulation accelerates nonviral direct conversion of functional induced neuronal cells from fibroblasts, increases the conversion efficiency, and induces highly matured neuronal phenotypes with improved electrophysiological functionalities. TENG devices may also be used for biomedical in vivo reprogramming.

  11. Functional Multiple-Set Canonical Correlation Analysis

    ERIC Educational Resources Information Center

    Hwang, Heungsun; Jung, Kwanghee; Takane, Yoshio; Woodward, Todd S.

    2012-01-01

    We propose functional multiple-set canonical correlation analysis for exploring associations among multiple sets of functions. The proposed method includes functional canonical correlation analysis as a special case when only two sets of functions are considered. As in classical multiple-set canonical correlation analysis, computationally, the…

  12. Non invasive in vivo investigation of hepatobiliary structure and function in STII medaka (Oryzias latipes): methodology and applications

    PubMed Central

    Hardman, Ron C; Kullman, Seth W; Hinton, David E

    2008-01-01

    Background A novel transparent stock of medaka (Oryzias latipes; STII), recessive for all pigments found in chromatophores, permits transcutaneous imaging of internal organs and tissues in living individuals. Findings presented describe the development of methodologies for non invasive in vivo investigation in STII medaka, and the successful application of these methodologies to in vivo study of hepatobiliary structure, function, and xenobiotic response, in both 2 and 3 dimensions. Results Using brightfield, and widefield and confocal fluorescence microscopy, coupled with the in vivo application of fluorescent probes, structural and functional features of the hepatobiliary system, and xenobiotic induced toxicity, were imaged at the cellular level, with high resolution (< 1 μm), in living individuals. The findings presented demonstrate; (1) phenotypic response to xenobiotic exposure can be investigated/imaged in vivo with high resolution (< 1 μm), (2) hepatobiliary transport of solutes from blood to bile can be qualitatively and quantitatively studied/imaged in vivo, (3) hepatobiliary architecture in this lower vertebrate liver can be studied in 3 dimensions, and (4) non invasive in vivo imaging/description of hepatobiliary development in this model can be investigated. Conclusion The non-invasive in vivo methodologies described are a unique means by which to investigate biological structure, function and xenobiotic response with high resolution in STII medaka. In vivo methodologies also provide the future opportunity to integrate molecular mechanisms (e.g., genomic, proteomic) of disease and toxicity with phenotypic changes at the cellular and system levels of biological organization. While our focus has been the hepatobiliary system, other organ systems are equally amenable to in vivo study, and we consider the potential for discovery, within the context of in vivo investigation in STII medaka, as significant. PMID:18838008

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

    PubMed

    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.

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

    PubMed Central

    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

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

    PubMed

    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.

  16. PET and SPECT Radiotracers to Assess Function and Expression of ABC Transporters in Vivo

    PubMed Central

    Mairinger, Severin; Erker, Thomas; Müller, Markus; Langer, Oliver

    2013-01-01

    Adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated proteins (MRPs) are expressed in high concentrations at various physiological barriers (e.g. blood-brain barrier, blood-testis barrier, blood-tumor barrier), where they impede the tissue accumulation of various drugs by active efflux transport. Changes in ABC transporter expression and function are thought to be implicated in various diseases, such as cancer, epilepsy, Alzheimer’s and Parkinson’s disease. The availability of a non-invasive imaging method which allows for measuring ABC transporter function or expression in vivo would be of great clinical use in that it could facilitate the identification of those patients that would benefit from treatment with ABC transporter modulating drugs. To date three different kinds of imaging probes have been described to measure ABC transporters in vivo: i) radiolabelled transporter substrates ii) radiolabelled transporter inhibitors and iii) radiolabelled prodrugs which are enzymatically converted into transporter substrates in the organ of interest (e.g. brain). The design of new imaging probes to visualize efflux transporters is inter alia complicated by the overlapping substrate recognition pattern of different ABC transporter types. The present article will describe currently available ABC transporter radiotracers for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) and critically discuss strengths and limitations of individual probes and their potential clinical applications. PMID:21434859

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

    SciTech Connect

    Rouse, B.T.; Hartley, D.; Doherty, P.C. )

    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.

  18. High throughput in vivo functional validation of candidate congenital heart disease genes in Drosophila

    PubMed Central

    Zhu, Jun-yi; Fu, Yulong; Nettleton, Margaret; Richman, Adam; Han, Zhe

    2017-01-01

    Genomic sequencing has implicated large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system is needed to validate gene associations with pathology. We developed a Drosophila-based functional system to screen candidate disease genes identified from Congenital Heart Disease (CHD) patients. 134 genes were tested in the Drosophila heart using RNAi-based gene silencing. Quantitative analyses of multiple cardiac phenotypes demonstrated essential structural, functional, and developmental roles for more than 70 genes, including a subgroup encoding histone H3K4 modifying proteins. We also demonstrated the use of Drosophila to evaluate cardiac phenotypes resulting from specific, patient-derived alleles of candidate disease genes. We describe the first high throughput in vivo validation system to screen candidate disease genes identified from patients. This approach has the potential to facilitate development of precision medicine approaches for CHD and other diseases associated with genetic factors. DOI: http://dx.doi.org/10.7554/eLife.22617.001 PMID:28084990

  19. Zinc supplementation augments in vivo antitumor effect of chemotherapy by restoring p53 function.

    PubMed

    Margalit, Ofer; Simon, Amos J; Yakubov, Eduard; Puca, Rosa; Yosepovich, Ady; Avivi, Camila; Jacob-Hirsch, Jasmine; Gelernter, Ilana; Harmelin, Alon; Barshack, Iris; Rechavi, Gideon; D'Orazi, Gabriella; Givol, David; Amariglio, Ninette

    2012-08-15

    Activated p53 is necessary for tumor suppression. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of functional p53. HIPK2 modulates wild-type p53 activity toward proapoptotic transcription and tumor suppression by the phosphorylation of serine 46. Knock-down of HIPK2 interferes with tumor suppression and sensitivity to chemotherapy. Combined administration of adriamycin and zinc restores activity of misfolded p53 and enables the induction of its proapoptotic and tumor suppressor functions in vitro and in vivo. We therefore looked for a cancer model where HIPK2 expression is low. MMTV-neu transgenic mice overexpressing HER2/neu, develop mammary tumors at puberty with a long latency, showing very low expression of HIPK2. Here we show that whereas these tumors are resistant to adriamycin treatment, a combination of adriamycin and zinc suppresses tumor growth in vivo in these mice, an effect evidenced by the histological features of the mammary tumors. The combined treatment of adriamycin and zinc also restores wild-type p53 conformation and induces proapoptotic transcription activity. These findings may open up new possibilities for the treatment of human cancers via the combination of zinc with chemotherapeutic agents, for a selected group of patients expressing low levels of HIPK2, with an intact p53. In addition, HIPK2 may serve as a new biomarker for tumor aggressiveness.

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

    PubMed Central

    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

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

    PubMed

    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.

  2. The synthesis and in vivo assembly of functional antibodies in yeast

    NASA Astrophysics Data System (ADS)

    Wood, Clive R.; Boss, Michael A.; Kenten, John H.; Calvert, Jane E.; Roberts, Nicola A.; Emtage, J. Spencer

    1985-04-01

    The yeast Saccharomyces cerevisiae can synthesize, process and secrete higher eukaryotic proteins1-5. We have investigated the expression of immunoglobulin chains in yeast and demonstrate here (1) the synthesis, processing and secretion of light and heavy chains, (2) the glycosylation of heavy chain, (3) the intracellular localization of these foreign proteins by immunofluorescence, and (4) the detection of functional antibodies in cells co-expressing both chains. This may provide the basis of a microbial fermentation process for the production of monoclonal antibodies. The co-expression of light and heavy chains in Escherichia coli has been reported but functional antibodies were not assembled in vivo6,7. Furthermore, only low-level assembly of these chains was found in vitro.

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

    PubMed

    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.

  4. Predicting In Vivo Anti-Hepatofibrotic Drug Efficacy Based on In Vitro High-Content Analysis

    PubMed Central

    Zheng, Baixue; Tan, Looling; Mo, Xuejun; Yu, Weimiao; Wang, Yan; Tucker-Kellogg, Lisa; Welsch, Roy E.; So, Peter T. C.; Yu, Hanry

    2011-01-01

    Background/Aims Many anti-fibrotic drugs with high in vitro efficacies fail to produce significant effects in vivo. The aim of this work is to use a statistical approach to design a numerical predictor that correlates better with in vivo outcomes. Methods High-content analysis (HCA) was performed with 49 drugs on hepatic stellate cells (HSCs) LX-2 stained with 10 fibrotic markers. ∼0.3 billion feature values from all cells in >150,000 images were quantified to reflect the drug effects. A systematic literature search on the in vivo effects of all 49 drugs on hepatofibrotic rats yields 28 papers with histological scores. The in vivo and in vitro datasets were used to compute a single efficacy predictor (Epredict). Results We used in vivo data from one context (CCl4 rats with drug treatments) to optimize the computation of Epredict. This optimized relationship was independently validated using in vivo data from two different contexts (treatment of DMN rats and prevention of CCl4 induction). A linear in vitro-in vivo correlation was consistently observed in all the three contexts. We used Epredict values to cluster drugs according to efficacy; and found that high-efficacy drugs tended to target proliferation, apoptosis and contractility of HSCs. Conclusions The Epredict statistic, based on a prioritized combination of in vitro features, provides a better correlation between in vitro and in vivo drug response than any of the traditional in vitro markers considered. PMID:22073152

  5. Functional surface engineering of C-dots for fluorescent biosensing and in vivo bioimaging.

    PubMed

    Ding, Changqin; Zhu, Anwei; Tian, Yang

    2014-01-21

    Nanoparticles are promising scaffolds for applications such as imaging, chemical sensors and biosensors, diagnostics, drug delivery, catalysis, energy, photonics, medicine, and more. Surface functionalization of nanoparticles introduces an additional dimension in controlling nanoparticle interfacial properties and provides an effective bridge to connect nanoparticles to biological systems. With fascinating photoluminescence properties, carbon dots (C-dots), carbon-containing nanoparticles that are attracting considerable attention as a new type of quantum dot, are becoming both an important class of imaging probes and a versatile platform for engineering multifunctional nanosensors. In order to transfer C-dots from proof-of-concept studies toward real world applications such as in vivo bioimaging and biosensing, careful design and engineering of C-dot probes is becoming increasingly important. A comprehensive knowledge of how C-dot surfaces with various properties behave is essential for engineering C-dots with useful imaging properties such as high quantum yield, stability, and low toxicity, and with desirable biosensing properties such as high selectivity, sensitivity, and accuracy. Several reviews in recent years have reported preparation methods and properties of C-dots and described their application in biosensors, catalysis, photovoltatic cells, and more. However, no one has yet systematically summarized the surface engineering of C-dots, nor the use of C-dots as fluorescent nanosensors or probes for in vivo imaging in cells, tissues, and living organisms. In this Account, we discuss the major design principles and criteria for engineering the surface functionality of C-dots for biological applications. These criteria include brightness, long-term stability, and good biocompatibility. We review recent developments in designing C-dot surfaces with various functionalities for use as nanosensors or as fluorescent probes with fascinating analytical performance

  6. Protective effects of Zhuyeqing liquor on the immune function of normal and immunosuppressed mice in vivo

    PubMed Central

    2013-01-01

    Background Zhuyeqing Liquor (ZYQL), a well-known Chinese traditional health liquor, has various biological properties, including anti-oxidant, anti-inflammatory, immunoenhancement and cardiovascular protective effects. Methods The protective effects of Zhuyeqing Liquor (ZYQL) on the immune function was investigated in vivo in normal healthy mice and immunosuppressed mice treated with Cyclophosphamide (Cy, 100 mg/kg) by intraperitoneal injection on days 4, 8 and 12. ZYQL (100, 200 and 400 mg/kg) was administered via gavage daily for 14 days. The phagocytotic function of mononuclear phagocytic system was detected with carbon clearance methods, the levels of interleukin-6 (IL-6) and interferon-gamma (IFN-γ) in serum were detected with Enzyme linked immunosorbent assay (ELISA). Immune organs were weighed and organ indexes (organ weight/body weight) of thymus and spleen were calculated. Meanwhile, the activity of lysozyme (LSZ) in serum and the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) in spleen tissue were measured. Results ZYQL significantly upgrades the K value for clearance of carbon particles in normal mice treated with ZYQL (400 mg/kg) and immunosuppressed mice treated with ZYQL (100, 200 and 400 mg/kg) together with Cy (100 mg/kg) in vivo. The treatment of ZYQL (100, 200 and 400 mg/kg) effectively increased the activity of serum lysozyme as well as promoted the serum levels of IL-6 and IFN-γ in normal mice and immunosuppressed mice. Furthermore, ZYQL (100, 200 and 400 mg/kg) had an antioxidant effects in immune system by enhancing the antioxidant enzyme activity of SOD, CAT and GSH-Px in vivo. In addition, ZYQL (100, 200 and 400 mg/kg) effectively elevated the Cy-induced decreased organ index (thymus and spleen). Conclusions The present work shows that the dose-dependent administration of ZYQL is capable of influencing immune responses, which implying that its valuable functional health may be attributed

  7. In vivo relationship between pelvis motion and deep fascia displacement of the medial gastrocnemius: anatomical and functional implications.

    PubMed

    Cruz-Montecinos, Carlos; González Blanche, Alberto; López Sánchez, David; Cerda, Mauricio; Sanzana-Cuche, Rodolfo; Cuesta-Vargas, Antonio

    2015-11-01

    Different authors have modelled myofascial tissue connectivity over a distance using cadaveric models, but in vivo models are scarce. The aim of this study was to evaluate the relationship between pelvic motion and deep fascia displacement in the medial gastrocnemius (MG). Deep fascia displacement of the MG was evaluated through automatic tracking with an ultrasound. Angular variation of the pelvis was determined by 2D kinematic analysis. The average maximum fascia displacement and pelvic motion were 1.501 ± 0.78 mm and 6.55 ± 2.47 °, respectively. The result of a simple linear regression between fascia displacement and pelvic motion for three task executions by 17 individuals was r = 0.791 (P < 0.001). Moreover, hamstring flexibility was related to a lower anterior tilt of the pelvis (r = 0.544, P < 0.024) and a lower deep fascia displacement of the MG (r = 0.449, P < 0.042). These results support the concept of myofascial tissue connectivity over a distance in an in vivo model, reinforce the functional concept of force transmission through synergistic muscle groups, and grant new perspectives for the role of fasciae in restricting movement in remote zones.

  8. On the origin and functions of the term functional analysis.

    PubMed

    Schlinger, Henry D; Normand, Matthew P

    2013-01-01

    In this essay, we note that although Iwata, Dorsey, Slifer, Bauman, and Richman (1982) established the standard framework for conducting functional analyses of problem behavior, the term functional analysis was probably first used in behavior analysis by B. F. Skinner in 1948. We also remind readers that a functional analysis is really an experimental analysis, words that were contained in the title of Skinner's first book, The Behavior of Organisms: An Experimental Analysis (1938). We further describe how Skinner initially applied the concept of functional analysis to an understanding of verbal behavior, and we suggest that the same tactic be applied to the verbal behavior of behavior analysts, in the present case, to the term functional analysis.

  9. Body adiposity dictates different mechanisms of increased coronary reactivity related to improved in vivo cardiac function

    PubMed Central

    2014-01-01

    Background Saturated fatty acid-rich high fat (HF) diets trigger abdominal adiposity, insulin resistance, type 2 diabetes and cardiac dysfunction. This study was aimed at evaluating the effects of nascent obesity on the cardiac function of animals fed a high-fat diet and at analyzing the mechanisms by which these alterations occurred at the level of coronary reserve. Materials and methods Rats were fed a control (C) or a HF diet containing high proportions of saturated fatty acids for 3 months. Thereafter, their cardiac function was evaluated in vivo using a pressure probe inserted into the cavity of the left ventricle. Their heart was isolated, perfused iso-volumetrically according to the Langendorff mode and the coronary reserve was evaluated by determining the endothelial-dependent (EDV) and endothelial-independent (EIV) vasodilatations in the absence and presence of endothelial nitric oxide synthase and cyclooxygenase inhibitors (L-NAME and indomethacin). The fatty acid composition of cardiac phospholipids was then evaluated. Results Although all the HF-fed rats increased their abdominal adiposity, some of them did not gain body weight (HF- group) compared to the C group whereas other ones had a higher body weight (HF+). All HF rats displayed a higher in vivo cardiac activity associated with an increased EDV. In the HF- group, the improved EDV was due to an increase in the endothelial cell vasodilatation activity whereas in the HF+ group, the enhanced EDV resulted from an improved sensitivity of coronary smooth muscle cells to nitric oxide. Furthermore, in the HF- group the main pathway implicated in the EDV was the NOS pathway while in the HF+ group the COX pathway. Conclusions Nascent obesity-induced improvement of cardiac function may be supported by an enhanced coronary reserve occurring via different mechanisms. These mechanisms implicate either the endothelial cells activity or the smooth muscle cells sensitivity depending on the body adiposity of

  10. Selective ex-vivo photothermal ablation of human pancreatic cancer with albumin functionalized multiwalled carbon nanotubes

    PubMed Central

    Mocan, Lucian; Tabaran, Flaviu A; Mocan, Teodora; Bele, Constantin; Orza, Anamaria Ioana; Lucan, Ciprian; Stiufiuc, Rares; Manaila, Ioana; Iulia, Ferencz; Dana, Iancu; Zaharie, Florin; Osian, Gelu; Vlad, Liviu; Iancu, Cornel

    2011-01-01

    The process of laser-mediated ablation of cancer cells marked with biofunctionalized carbon nanotubes is frequently called “nanophotothermolysis”. We herein present a method of selective nanophotothermolisys of pancreatic cancer (PC) using multiwalled carbon nanotubes (MWCNTs) functionalized with human serum albumin (HSA). With the purpose of testing the therapeutic value of these nanobioconjugates, we have developed an ex-vivo experimental platform. Surgically resected specimens from patients with PC were preserved in a cold medium and kept alive via intra-arterial perfusion. Additionally, the HSA-MWCNTs have been intra-arterially administered in the greater pancreatic artery under ultrasound guidance. Confocal and transmission electron microscopy combined with immunohistochemical staining have confirmed the selective accumulation of HSA-MWCNTs inside the human PC tissue. The external laser irradiation of the specimen has significantly produced extensive necrosis of the malign tissue after the intra-arterial administration of HSA-MWCNTs, without any harmful effects on the surrounding healthy parenchyma. We have obtained a selective photothermal ablation of the malign tissue based on the selective internalization of MWCNTs with HSA cargo inside the pancreatic adenocarcinoma after the ex-vivo intra-arterial perfusion. PMID:21720504

  11. Head-to-tail regulation is critical for the in vivo function of myosin V

    PubMed Central

    Donovan, Kirk W.

    2015-01-01

    Cell organization requires regulated cargo transport along cytoskeletal elements. Myosin V motors are among the most conserved organelle motors and have been well characterized in both yeast and mammalian systems. Biochemical data for mammalian myosin V suggest that a head-to-tail autoinhibitory interaction is a primary means of regulation, but the in vivo significance of this interaction has not been studied. Here we generated and characterized mutations in the yeast myosin V Myo2p to reveal that it is regulated by a head-to-tail interaction and that loss of regulation renders the myosin V constitutively active. We show that an unregulated motor is very deleterious for growth, resulting in severe defects in Myo2-mediated transport processes, including secretory vesicle transport, mitochondrial inheritance, and nuclear orientation. All of the defects associated with motor misregulation could be rescued by artificially restoring regulation. Thus, spatial and temporal regulation of myosin V in vivo by a head-to-tail interaction is critical for the normal delivery functions of the motor. PMID:25940346

  12. Structure and function of RNase AS, a polyadenylate-specific exoribonuclease affecting mycobacterial virulence in vivo.

    PubMed

    Romano, Maria; van de Weerd, Robert; Brouwer, Femke C C; Roviello, Giovanni N; Lacroix, Ruben; Sparrius, Marion; van den Brink-van Stempvoort, Gunny; Maaskant, Janneke J; van der Sar, Astrid M; Appelmelk, Ben J; Geurtsen, Jeroen J; Berisio, Rita

    2014-05-06

    The cell-envelope of Mycobacterium tuberculosis plays a key role in bacterial virulence and antibiotic resistance. Little is known about the molecular mechanisms of regulation of cell-envelope formation. Here, we elucidate functional and structural properties of RNase AS, which modulates M. tuberculosis cell-envelope properties and strongly impacts bacterial virulence in vivo. The structure of RNase AS reveals a resemblance to RNase T from Escherichia coli, an RNase of the DEDD family involved in RNA maturation. We show that RNase AS acts as a 3'-5'-exoribonuclease that specifically hydrolyzes adenylate-containing RNA sequences. Also, crystal structures of complexes with AMP and UMP reveal the structural basis for the observed enzyme specificity. Notably, RNase AS shows a mechanism of substrate recruitment, based on the recognition of the hydrogen bond donor NH2 group of adenine. Our work opens a field for the design of drugs able to reduce bacterial virulence in vivo.

  13. In vivo ultrasound imaging of the popliteus muscle: investigation of functional characteristics

    PubMed Central

    Soda, Naoki; Fujihashi, Yuichiro; Aoki, Takaaki

    2016-01-01

    [Purpose] The aim of this study was to use ultrasound imaging equipment for in vivo observation of the popliteus muscle thickness during rest and exercise to examine its functional characteristics and to establish a training method for this muscle. [Subjects and Methods] The subjects included 30 healthy adults (15 men and 15 women). The measurement tasks, consisting of isometric knee flexion and extension and internal rotation of the lower leg were performed in an arbitrary order. The popliteus muscle thickness was measured using an ultrasound. [Results] The popliteus muscle thickness significantly increased in the internal rotation in 27 subjects (90%), whereas, it remained unchanged in the remaining three subjects (10%). [Conclusion] This study differed from most of the previous studies because it involved in vivo observation of the popliteus muscle. We found that ultrasound was an effective method for the measurement of popliteus muscle thickness. The results suggest that internal rotation of the lower leg is the most effective exercise for working the popliteus muscle. PMID:27134397

  14. An intramolecular signaling element that modulates dynamin function in vitro and in vivo.

    PubMed

    Chappie, Joshua S; Acharya, Sharmistha; Liu, Ya-Wen; Leonard, Marilyn; Pucadyil, Thomas J; Schmid, Sandra L

    2009-08-01

    Dynamin exhibits a high basal rate of GTP hydrolysis that is enhanced by self-assembly on a lipid template. Dynamin's GTPase effector domain (GED) is required for this stimulation, though its mechanism of action is poorly understood. Recent structural work has suggested that GED may physically dock with the GTPase domain to exert its stimulatory effects. To examine how these interactions activate dynamin, we engineered a minimal GTPase-GED fusion protein (GG) that reconstitutes dynamin's basal GTPase activity and utilized it to define the structural framework that mediates GED's association with the GTPase domain. Chemical cross-linking of GG and mutagenesis of full-length dynamin establishes that the GTPase-GED interface is comprised of the N- and C-terminal helices of the GTPase domain and the C-terminus of GED. We further show that this interface is essential for structural stability in full-length dynamin. Finally, we identify mutations in this interface that disrupt assembly-stimulated GTP hydrolysis and dynamin-catalyzed membrane fission in vitro and impair the late stages of clathrin-mediated endocytosis in vivo. These data suggest that the components of the GTPase-GED interface act as an intramolecular signaling module, which we term the bundle signaling element, that can modulate dynamin function in vitro and in vivo.

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

    PubMed

    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.

  16. Physiologically inspired cardiac scaffolds for tailored in vivo function and heart regeneration

    PubMed Central

    Kaiser, Nicholas J; Coulombe, Kareen L K

    2015-01-01

    Tissue engineering is well suited for the treatment of cardiac disease due to the limited regenerative capacity of native cardiac tissue and the loss of function associated with endemic cardiac pathologies, such as myocardial infarction and congenital heart defects. However, the physiological complexity of the myocardium imposes extensive requirements on tissue therapies intended for these applications. In recent years, the field of cardiac tissue engineering has been characterized by great innovation and diversity in the fabrication of engineered tissue scaffolds for cardiac repair and regeneration to address these problems. From early approaches that attempted only to deliver cardiac cells in a hydrogel vessel, significant progress has been made in understanding the role of each major component of cardiac living tissue constructs (namely cells, scaffolds, and signaling mechanisms) as they relate to mechanical, biological, and electrical in vivo performance. This improved insight, accompanied by modern material science techniques, allows for the informed development of complex scaffold materials that are optimally designed for cardiac applications. This review provides a background on cardiac physiology as it relates to critical cardiac scaffold characteristics, the degree to which common cardiac scaffold materials fulfill these criteria, and finally an overview of recent in vivo studies that have employed this type of approach. PMID:25970645

  17. In Vivo Evaluation of Vena Caval Filters: Can Function Be Linked to Design Characteristics?

    SciTech Connect

    Proctor, Mary C.; Cho, Kyung J.; Greenfield, Lazar J.

    2000-11-15

    Purpose: To compare the five vena caval filters marketed in the United States and one investigational vena caval filter and to determine whether there is an association between their design and their in vivo function.Methods: Four of each type of filter-Simon Nitinol (SN), Bird's Nest (BN), Vena Tech (VT), Greenfield stainless steel (PSGF), Greenfield titanium (TGF), and the investigational stent cone filter (NGF)-were studied for 60 days in 12 sheep. Radiographic and pathologic outcomes to be assessed included clot capture and resolution, vena caval penetration, position of the filter, thrombogenicity, and vessel wall reaction.Results: Filters differed with respect to the number of clot-trapping levels and the interdependence of the legs. All devices were successfully placed. Intentionally embolized clot was captured. One VT and two SN filters migrated in response to clot capture. Resolution of thrombus was variable, and related to the design of the device. Fibrin webbing was widely present with the VT, BN, and SN filters but limited in the others. The VT and NGF filters demonstrated the most stable filter base diameter.Conclusions: The performance of vena caval filters differs with respect to clot resolution and mechanical stability. Interdependent filter limbs and single-stage conical capture sites appear to result in more favorable performance in in vivo studies.

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

    PubMed Central

    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

  19. In vivo estimation of the glenohumeral joint centre by functional methods: accuracy and repeatability assessment.

    PubMed

    Lempereur, Mathieu; Leboeuf, Fabien; Brochard, Sylvain; Rousset, Jean; Burdin, Valérie; Rémy-Néris, Olivier

    2010-01-19

    Several algorithms have been proposed for determining the centre of rotation of ball joints. These algorithms are used rather to locate the hip joint centre. Few studies have focused on the determination of the glenohumeral joint centre. However, no studies have assessed the accuracy and repeatability of functional methods for glenohumeral joint centre. This paper aims at evaluating the accuracy and the repeatability with which the glenohumeral joint rotation centre (GHRC) can be estimated in vivo by functional methods. The reference joint centre is the glenohumeral anatomical centre obtained by medical imaging. Five functional methods were tested: the algorithm of Gamage and Lasenby (2002), bias compensated (Halvorsen, 2003), symmetrical centre of rotation estimation (Ehrig et al., 2006), normalization method (Chang and Pollard, 2007), helical axis (Woltring et al., 1985). The glenohumeral anatomical centre (GHAC) was deduced from the fitting of the humeral head. Four subjects performed three cycles of three different movements (flexion/extension, abduction/adduction and circumduction). For each test, the location of the glenohumeral joint centre was estimated by the five methods. Analyses focused on the 3D location, on the repeatability of location and on the accuracy by computing the Euclidian distance between the estimated GHRC and the GHAC. For all the methods, the error repeatability was inferior to 8.25 mm. This study showed that there are significant differences between the five functional methods. The smallest distance between the estimated joint centre and the centre of the humeral head was obtained with the method of Gamage and Lasenby (2002).

  20. Functional Analysis and Treatment of Nail Biting

    ERIC Educational Resources Information Center

    Dufrene, Brad A.; Watson, T. Steuart; Kazmerski, Jennifer S.

    2008-01-01

    This study applied functional analysis methodology to nail biting exhibited by a 24-year-old female graduate student. Results from the brief functional analysis indicated variability in nail biting across assessment conditions. Functional analysis data were then used to guide treatment development and implementation. Treatment included a…

  1. In Vivo Analysis of the Major Exocytosis-sensitive Phosphoprotein in Tetrahymena

    PubMed Central

    Chilcoat, N. Doane; Turkewitz, Aaron P.

    1997-01-01

    Phosphoglucomutase (PGM) is a ubiquitous highly conserved enzyme involved in carbohydrate metabolism. A number of recently discovered PGM-like proteins in a variety of organisms have been proposed to function in processes other than metabolism. In addition, sequence analysis suggests that several of these may lack PGM enzymatic activity. The best studied PGM-like protein is parafusin, a major phosphoprotein in the ciliate Paramecium tetraurelia that undergoes rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules. Indirect genetic and biochemical evidence also supports a role in regulated exocytotic membrane fusion. To examine this matter directly, we have identified and cloned the parafusin homologue in Tetrahymena thermophila, a ciliate in which protein function can be studied in vivo. The unique T. thermophila gene, called PGM1, encodes a protein that is closely related to parafusin by sequence and by characteristic post-translational modifications. Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity. We evaluated the activity and function of PGM1 through gene disruption. Surprisingly, ΔPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity. Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion. PMID:9382866

  2. Clinical applications of in vivo neutron-activation analysis

    SciTech Connect

    Cohn, S.H.

    1982-01-01

    In vivo neutron activation has opened a new era of both clinical diagnosis and therapy evaluation, and investigation into and modelling of body composition. The techniques are new, but it is already clear that considerable strides can be made in increasing accuracy and precision, increasing the number of elements susceptible to measurement, enhancing uniformity, and reducing the dose required for the measurement. The work presently underway will yield significant data on a variety of environmental contaminants such as Cd. Compositional studies are determining the level of vital constituents such as nitrogen and potassium in both normal subjects and in patients with a variety of metabolic disorders. Therapeutic programs can be assessed while in progress.

  3. In-vivo neutron activation analysis: principles and clinical applications

    SciTech Connect

    Cohn, S.H.

    1982-01-01

    In vivo neutron activation has opened a new era of both clinical diagnosis and therapy evaluation, and investigation into and modelling of body composition. The techniques are new, but it is already clear that considerable strides can be made in increasing accuracy and precision, increasing the number of elements susceptible to measurement, enhancing uniformity, and reducing the dose required for the measurement. The work presently underway will yield significant data on a variety of environmental contaminants such as Cd. Compositional studies are determining the level of vital constituents such as nitrogen and potassium in both normal subjects and in patients with a variety of metabolic disorders. Therapeutic programs can be assessed while in progress. It seems likely that by the end of this century there will have been significant progress with this research tool, and exciting insights obtained into the nature and dynamics of human body composition.

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

    PubMed

    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.

  5. Optical detection of brain function: simultaneous imaging of cerebral vascular response, tissue metabolism, and cellular activity in vivo.

    PubMed

    Du, Congwu; Pan, Yingtian

    2011-01-01

    It is known that a remaining challenge for functional brain imaging is to distinguish the coupling and decoupling effects among neuronal activity, cerebral metabolism, and vascular hemodynamics, which highlights the need for new tools to enable simultaneous measures of these three properties in vivo. Here, we review current neuroimaging techniques and their prospects and potential limitations for tackling this challenge. We then report a novel dual-wavelength laser speckle imaging (DW-LSI) tool developed in our labs that enables simultaneous imaging of cerebral blood flow (CBF), cerebral blood volume, and tissue hemoglobin oxygenation, which allows us to monitor neurovascular and tissue metabolic activities at high spatiotemporal resolutions over a relatively large field of view. Moreover, we report digital frequency ramping Doppler optical coherence tomography (DFR-OCT) that allows for quantitative 3D imaging of the CBF network in vivo. In parallel, we review calcium imaging techniques to track neuronal activity, including intracellular calcium approach using Rhod2 fluorescence technique that we develop to detect neuronal activity in vivo. We report a new multimodality imaging platform that combines DW-LSI, DFR-OCT, and calcium fluorescence imaging for simultaneous detection of cortical hemodynamics, cerebral metabolism, and neuronal activities of the animal brain in vivo, as well as its integration with microprobes for imaging neuronal function in deep brain regions in vivo. Promising results of in vivo animal brain functional studies suggest the potential of this multimodality approach for future awake animal and behavioral studies.

  6. In Vivo Imaging Sheds Light on Immune Cell Migration and Function in Cancer

    PubMed Central

    Torcellan, Tommaso; Stolp, Jessica; Chtanova, Tatyana

    2017-01-01

    There is ample evidence for both beneficial and harmful involvement of the immune system in tumor development and spread. Immune cell recruitment to tumors is essential not only for the success of anticancer immune therapies but also for tumor-induced immune suppression. Now that immune-based therapies are playing an increasingly important role in treatment of solid tumors such as metastatic melanomas, precise analysis of the in vivo contributions of different leukocyte subsets in tumor immunity has become an even greater priority. Recently, this goal has been markedly facilitated by the use of intravital microscopy, which has enabled us to visualize the dynamic interactions between cells of the immune system and tumor targets in the context of the tumor microenvironment. For example, intravital imaging techniques have shed new light on T cell infiltration of tumors, the mechanisms of cancer cell killing, and how myeloid cells contribute to tumor tolerance and spread. This mini-review summarizes the recent advances made to our understanding of the roles of innate and adaptive immune cells in cancer based on the use of these in vivo imaging approaches. PMID:28382036

  7. In Vivo Imaging Sheds Light on Immune Cell Migration and Function in Cancer.

    PubMed

    Torcellan, Tommaso; Stolp, Jessica; Chtanova, Tatyana

    2017-01-01

    There is ample evidence for both beneficial and harmful involvement of the immune system in tumor development and spread. Immune cell recruitment to tumors is essential not only for the success of anticancer immune therapies but also for tumor-induced immune suppression. Now that immune-based therapies are playing an increasingly important role in treatment of solid tumors such as metastatic melanomas, precise analysis of the in vivo contributions of different leukocyte subsets in tumor immunity has become an even greater priority. Recently, this goal has been markedly facilitated by the use of intravital microscopy, which has enabled us to visualize the dynamic interactions between cells of the immune system and tumor targets in the context of the tumor microenvironment. For example, intravital imaging techniques have shed new light on T cell infiltration of tumors, the mechanisms of cancer cell killing, and how myeloid cells contribute to tumor tolerance and spread. This mini-review summarizes the recent advances made to our understanding of the roles of innate and adaptive immune cells in cancer based on the use of these in vivo imaging approaches.

  8. In vivo functions of the gamma-butyrolactone autoregulator receptor in Streptomyces ambofaciens producing spiramycin.

    PubMed

    Choi, Sun-Uk; Kim, Mi-Kyung; Ha, Heon-Su; Hwang, Yong-Il

    2008-05-01

    A gene encoding a gamma-butyrolactone autoregulator receptor was cloned in to E. coli from Streptomyces ambofaciens producing spiramycin, a macrolide antibiotic used in both veterinary medicine and human medicine. A 714-bp intact receptor gene (saaR) was obtained by PCR and genomic Southern hybridization with the 100-bp PCR product as a probe. To clarify the in vivo function of saaR, a saaR-disrupted strain was constructed by means of homologous recombination, and phenotypes were compared with those of the wild-type strain. The number of saaR-disruptant spores was 4-fold less than that of the wild-type strain. In addition, saaR deletion from the S. ambofaciens chromosome resulted in complete loss of spiramycin production suggesting that saaR is a rare positive regulator, controlling both spiramycin biosynthesis and sporulation.

  9. Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets.

    PubMed

    Ohashi, Kazuo; Yokoyama, Takashi; Yamato, Masayuki; Kuge, Hiroyuki; Kanehiro, Hiromichi; Tsutsumi, Masahiro; Amanuma, Toshihiro; Iwata, Hiroo; Yang, Joseph; Okano, Teruo; Nakajima, Yoshiyuki

    2007-07-01

    Hepatic tissue engineering using primary hepatocytes has been considered a valuable new therapeutic modality for several classes of liver diseases. Recent progress in the development of clinically feasible liver tissue engineering approaches, however, has been hampered mainly by insufficient cell-to-cell contact of the engrafted hepatocytes. We developed a method to engineer a uniformly continuous sheet of hepatic tissue using isolated primary hepatocytes cultured on temperature-responsive surfaces. Sheets of hepatic tissue transplanted into the subcutaneous space resulted in efficient engraftment to the surrounding cells, with the formation of two-dimensional hepatic tissues that stably persisted for longer than 200 d. The engineered hepatic tissues also showed several characteristics of liver-specific functionality. Additionally, when the hepatic tissue sheets were layered in vivo, three-dimensional miniature liver systems having persistent survivability could be also engineered. This technology for liver tissue engineering is simple, minimally invasive and free of potentially immunogenic biodegradable scaffolds.

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

    PubMed

    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.

  11. In vivo function and comparative genomic analyses of the Drosophila gut microbiota identify candidate symbiosis factors

    PubMed Central

    Newell, Peter D.; Chaston, John M.; Wang, Yiping; Winans, Nathan J.; Sannino, David R.; Wong, Adam C. N.; Dobson, Adam J.; Kagle, Jeanne; Douglas, Angela E.

    2014-01-01

    Symbiosis is often characterized by co-evolutionary changes in the genomes of the partners involved. An understanding of these changes can provide insight into the nature of the relationship, including the mechanisms that initiate and maintain an association between organisms. In this study we examined the genome sequences of bacteria isolated from the Drosophila melanogaster gut with the objective of identifying genes that are important for function in the host. We compared microbiota isolates with con-specific or closely related bacterial species isolated from non-fly environments. First the phenotype of germ-free Drosophila (axenic flies) was compared to that of flies colonized with specific bacteria (gnotobiotic flies) as a measure of symbiotic function. Non-fly isolates were functionally distinct from bacteria isolated from flies, conferring slower development and an altered nutrient profile in the host, traits known to be microbiota-dependent. Comparative genomic methods were next employed to identify putative symbiosis factors: genes found in bacteria that restore microbiota-dependent traits to gnotobiotic flies, but absent from those that do not. Factors identified include riboflavin synthesis and stress resistance. We also used a phylogenomic approach to identify protein coding genes for which fly-isolate sequences were more similar to each other than to other sequences, reasoning that these genes may have a shared function unique to the fly environment. This method identified genes in Acetobacter species that cluster in two distinct genomic loci: one predicted to be involved in oxidative stress detoxification and another encoding an efflux pump. In summary, we leveraged genomic and in vivo functional comparisons to identify candidate traits that distinguish symbiotic bacteria. These candidates can serve as the basis for further work investigating the genetic requirements of bacteria for function and persistence in the Drosophila gut. PMID:25408687

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

    PubMed

    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.

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

    PubMed

    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.

  14. Artemisia scoparia Enhances Adipocyte Development and Endocrine Function In Vitro and Enhances Insulin Action In Vivo

    PubMed Central

    Richard, Allison J.; Fuller, Scott; Fedorcenco, Veaceslav; Beyl, Robbie; Burris, Thomas P.; Mynatt, Randall; Ribnicky, David M.; Stephens, Jacqueline M.

    2014-01-01

    Background Failure of adipocytes to expand during periods of energy excess can result in undesirable metabolic consequences such as ectopic fat accumulation and insulin resistance. Blinded screening studies have indicated that Artemisia scoparia (SCO) extracts can enhance adipocyte differentiation and lipid accumulation in cultured adipocytes. The present study tested the hypothesis that SCO treatment modulates fat cell development and function in vitro and insulin sensitivity in adipose tissue in vivo. Methods In vitro experiments utilized a Gal4-PPARγ ligand binding domain (LBD) fusion protein-luciferase reporter assay to examine PPARγ activation. To investigate the ability of SCO to modulate adipogenesis and mature fat cell function in 3T3-L1 cells, neutral lipid accumulation, gene expression, and protein secretion were measured by Oil Red O staining, qRT-PCR, and immunoblotting, respectively. For the in vivo experiments, diet-induced obese (DIO) C57BL/6J mice were fed a high-fat diet (HFD) or HFD containing 1% w/w SCO for four weeks. Body weight and composition, food intake, and fasting glucose and insulin levels were measured. Phospho-activation and expression of insulin-sensitizing proteins in epididymal adipose tissue (eWAT) were measured by immunoblotting. Results Ethanolic extracts of A. scoparia significantly activated the PPARγ LBD and enhanced lipid accumulation in differentiating 3T3-L1 cells. SCO increased the transcription of several PPARγ target genes in differentiating 3T3-L1 cells and rescued the negative effects of tumor necrosis factor α on production and secretion of adiponectin and monocyte chemoattractant protein-1 in fully differentiated fat cells. DIO mice treated with SCO had elevated adiponectin levels and increased phosphorylation of AMPKα in eWAT when compared to control mice. In SCO-treated mice, these changes were also associated with decreased fasting insulin and glucose levels. Conclusion SCO has metabolically beneficial

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

    SciTech Connect

    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

  16. In Vitro and In Vivo Evaluation of a Novel Ferrocyanide Functionalized Nanopourous Silica Decorporation Agent for Cesium (Cs) in Rats

    PubMed Central

    Timchalk, Charles; Creim, Jeffrey A; Sukwarotwat, Vichaya; Wiacek, Robert; Addleman, R Shane; Fryxell, Glen E; Yantasee, Wassana

    2009-01-01

    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); therefore 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. Animals in group I were administered 137Cs chloride (~40 μg/kg) by intravenous (iv) injection or oral gavage; Group II animals were administered pre-bound 137Cs- SAMMS or sequential 137Cs chloride + SAMMS (~61 ng/kg) by oral gavage; and Group III was orally administered 137Cs chloride (~61 ng/kg) followed by either 0.1 g of 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 chloride 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 outperforms Prussian blue in vitro at low pH, but

  17. Functional analysis and treatment of nail biting.

    PubMed

    Dufrene, Brad A; Steuart Watson, T; Kazmerski, Jennifer S

    2008-11-01

    This study applied functional analysis methodology to nail biting exhibited by a 24-year-old female graduate student. Results from the brief functional analysis indicated variability in nail biting across assessment conditions. Functional analysis data were then used to guide treatment development and implementation. Treatment included a simplified habit reversal package that was modified based on results of the functional analysis. Following treatment implementation, nail biting decreased as evidenced by consistent nail growth and participant self-recorded data. Results are discussed in terms of treatment utility of functional analysis methodology for novel populations and response topographies.

  18. Differential Item Functioning Analysis Using Rasch Item Information Functions

    ERIC Educational Resources Information Center

    Wyse, Adam E.; Mapuranga, Raymond

    2009-01-01

    Differential item functioning (DIF) analysis is a statistical technique used for ensuring the equity and fairness of educational assessments. This study formulates a new DIF analysis method using the information similarity index (ISI). ISI compares item information functions when data fits the Rasch model. Through simulations and an international…

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

    PubMed Central

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

    2016-01-01

    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. PMID:26987475

  20. In vivo imaging of mitochondrial function in methamphetamine-treated rats.

    PubMed

    Shiba, Takeshi; Yamato, Mayumi; Kudo, Wataru; Watanabe, Toshiaki; Utsumi, Hideo; Yamada, Ken-ichi

    2011-08-01

    Abuse of the powerfully addictive psychostimulant, methamphetamine, occurs worldwide. Recent studies have suggested that methamphetamine-induced dopaminergic neurotoxicity is related to oxidative stress. In response to nerve activation, the mitochondrial respiratory chain is rapidly activated. The enhancement of mitochondrial respiratory chain activation may induce oxidative stress in the brain. However, there is little experimental evidence regarding the mitochondrial function after methamphetamine administration in vivo. Here, we evaluated whether a single administration of methamphetamine induces ATP consumption and overactivation of mitochondria. We measured mitochondrial function in two different ways: by monitoring oxygen partial pressure using an oxygen-selective electrode, and by imaging of redox reactions using a nitroxyl radical (i.e., nitroxide) coupled with Overhauser-enhanced magnetic resonance imaging (OMRI). A single administration of methamphetamine to Wistar rats induced dopaminergic nerve activation, ATP consumption and an increase in mitochondrial respiratory chain function in both the striatum and cortex. Furthermore, antioxidant TEMPOL prevented the increase in mitochondrial oxidative damage and methamphetamine-induced sensitization. These findings suggest that energy-supplying reactions after dopaminergic nerve activation are associated with oxidative stress in both the striatum and cortex, leading to abnormal behavior.

  1. In vivo functional human imaging using photoacoustic microscopy: response to ischemic and thermal stimuli

    NASA Astrophysics Data System (ADS)

    Favazza, Christopher; Maslov, Konstantin; Cornelius, Lynn; Wang, Lihong V.

    2010-02-01

    We report results of two in vivo functional human imaging experiments using photoacoustic microscopy. In Experiment 1, the hemodynamic response to an ischemic event was measured. The palm of a volunteer was imaged and a single cross-section was monitored while periodic arterial occlusions were administered using a blood pressure cuff wrapped around the upper arm and inflated to ~280 mmHg. Significant relative decreases in oxygen saturation (sO2) and total hemoglobin (HbT) were observed during periods of ischemia. Upon release of the occlusion, significant relative increases in sO2 and HbT due to post-occlusive reactive hyperemia were recorded. Experiment 2 explored the vascular response to a local, external thermal stimulus. Thermal hyperemia is a common physiological phenomenon and thermoregulation function in which blood flow to the skin is increased to more efficiently exchange heat with the ambient environment. The forearm of a volunteer was imaged and a single cross-section was monitored while the imaged surface was exposed to an elevated temperature of ~46°C. Due to thermal hyperemia, relative increases in sO2 and HbT were measured as the temperature of the surface was raised. These results may contribute as clinically relevant measures of vascular functioning for detection and assessment of vascular related diseases.

  2. In Vivo Voltage-Sensitive Dye Imaging of Subcortical Brain Function.

    PubMed

    Tang, Qinggong; Tsytsarev, Vassiliy; Liang, Chia-Pin; Akkentli, Fatih; Erzurumlu, Reha S; Chen, Yu

    2015-11-27

    The whisker system of rodents is an excellent model to study peripherally evoked neural activity in the brain. Discrete neural modules represent each whisker in the somatosensory cortex ("barrels"), thalamus ("barreloids"), and brain stem ("barrelettes"). Stimulation of a single whisker evokes neural activity sequentially in its corresponding barrelette, barreloid, and barrel. Conventional optical imaging of functional activation in the brain is limited to surface structures such as the cerebral cortex. To access subcortical structures and image sensory-evoked neural activity, we designed a needle-based optical system using gradient-index (GRIN) rod lens. We performed voltage-sensitive dye imaging (VSDi) with GRIN rod lens to visualize neural activity evoked in the thalamic barreloids by deflection of whiskers in vivo. We stimulated several whiskers together to determine the sensitivity of our approach in differentiating between different barreloid responses. We also carried out stimulation of different whiskers at different times. Finally, we used muscimol in the barrel cortex to silence the corticothalamic inputs while imaging in the thalamus. Our results show that it is possible to obtain functional maps of the sensory periphery in deep brain structures such as the thalamic barreloids. Our approach can be broadly applicable to functional imaging of other core brain structures.

  3. In Vivo Voltage-Sensitive Dye Imaging of Subcortical Brain Function

    NASA Astrophysics Data System (ADS)

    Tang, Qinggong; Tsytsarev, Vassiliy; Liang, Chia-Pin; Akkentli, Fatih; Erzurumlu, Reha S.; Chen, Yu

    2015-11-01

    The whisker system of rodents is an excellent model to study peripherally evoked neural activity in the brain. Discrete neural modules represent each whisker in the somatosensory cortex (“barrels”), thalamus (“barreloids”), and brain stem (“barrelettes”). Stimulation of a single whisker evokes neural activity sequentially in its corresponding barrelette, barreloid, and barrel. Conventional optical imaging of functional activation in the brain is limited to surface structures such as the cerebral cortex. To access subcortical structures and image sensory-evoked neural activity, we designed a needle-based optical system using gradient-index (GRIN) rod lens. We performed voltage-sensitive dye imaging (VSDi) with GRIN rod lens to visualize neural activity evoked in the thalamic barreloids by deflection of whiskers in vivo. We stimulated several whiskers together to determine the sensitivity of our approach in differentiating between different barreloid responses. We also carried out stimulation of different whiskers at different times. Finally, we used muscimol in the barrel cortex to silence the corticothalamic inputs while imaging in the thalamus. Our results show that it is possible to obtain functional maps of the sensory periphery in deep brain structures such as the thalamic barreloids. Our approach can be broadly applicable to functional imaging of other core brain structures.

  4. Assessment of Ex Vivo Transport Function in Isolated Rodent Brain Capillaries.

    PubMed

    Chan, Gary N Y; Cannon, Ronald E

    2017-03-17

    The blood-brain barrier plays an important role in neuroprotection; however, it can be a major obstacle for drug delivery to the brain. This barrier primarily resides in the brain capillaries and functions as an interface between the brain and peripheral blood circulation. Several anatomical and biochemical elements of the blood-brain barrier are essential to regulate the permeability of nutrients, ions, hormones, toxic metabolites, and xenobiotics into and out of the brain. In particular, high expression of ATP-driven efflux transporters at the blood-brain barrier is a major obstacle in the delivery of CNS pharmacotherapeutics to the brain. The complete understanding of these elements can offer insights on how to modulate barrier functions for neuroprotection against CNS drug toxicity and to enhance drug delivery to the brain. In the literature, preclinical models of the blood-brain barrier are widely utilized to predict drug pharmacokinetics and pharmacodynamics properties in the brain. In addition, these models are essential tools to investigate cellular mechanisms and novel interventions that alter barrier function and permeability. This unit presents procedures to isolate fresh and viable rodent brain capillaries for the assessment of ex vivo transport activity at the blood-brain barrier. © 2017 by John Wiley & Sons, Inc.

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Blockade of CTLA-4 on CD4+CD25+ regulatory T cells abrogates their function in vivo.

    PubMed

    Read, Simon; Greenwald, Rebecca; Izcue, Ana; Robinson, Nicholas; Mandelbrot, Didier; Francisco, Loise; Sharpe, Arlene H; Powrie, Fiona

    2006-10-01

    Naturally occurring CD4+ regulatory T cells (T(R)) that express CD25 and the transcription factor FoxP3 play a key role in immune homeostasis, preventing immune pathological responses to self and foreign Ags. CTLA-4 is expressed by a high percentage of these cells, and is often considered as a marker for T(R) in experimental and clinical analysis. However, it has not yet been proven that CTLA-4 has a direct role in T(R) function. In this study, using a T cell-mediated colitis model, we demonstrate that anti-CTLA-4 mAb treatment inhibits T(R) function in vivo via direct effects on CTLA-4-expressing T(R), and not via hyperactivation of colitogenic effector T cells. Although anti-CTLA-4 mAb treatment completely inhibits T(R) function, it does not reduce T(R) numbers or their homing to the GALT, suggesting the Ab mediates its function by blockade of a signal required for T(R) activity. In contrast to the striking effect of the Ab, CTLA-4-deficient mice can produce functional T(R), suggesting that under some circumstances other immune regulatory mechanisms, including the production of IL-10, are able to compensate for the loss of the CTLA-4-mediated pathway. This study provides direct evidence that CTLA-4 has a specific, nonredundant role in the function of normal T(R). This role has to be taken into account when targeting CTLA-4 for therapeutic purposes, as such a strategy will not only boost effector T cell responses, but might also break T(R)-mediated self-tolerance.

  7. Increased osteoblast function in vitro and in vivo through surface nanostructuring by ultrasonic shot peening

    PubMed Central

    Guo, Yongyuan; Hu, Beibei; Tang, Chu; Wu, Yunpeng; Sun, Pengfei; Zhang, Xianlong; Jia, Yuhua

    2015-01-01

    Surface topography has significant influence on good and fast osseointegration of biomedical implants. In this work, ultrasonic shot peening was conducted to modify titanium to produce nanograined (NG) surface. Its ability to induce new bone formation was evaluated using an in vivo animal model. We demonstrated that the NG surface enhanced osteoblast adhesion, proliferation, differentiation, and mineralization in in vitro experiments compared to coarse-grained titanium surface. Push-out test, histological observations, fluorescent labeling, and histomorphometrical analysis consistently indicated that the NG surfaces developed have the higher osseointegration than coarse-grained surfaces. Those results suggest that ultrasonic shot peening has the potential for future use as a surface modification method in biomedical application. PMID:26229463

  8. Functional specificity of the homeodomain protein fushi tarazu: the role of DNA-binding specificity in vivo.

    PubMed Central

    Schier, A F; Gehring, W J

    1993-01-01

    The mechanisms determining the functional specificity of Drosophila homeodomain proteins are largely unknown. Here, the role of DNA-binding specificity for the in vivo function of the homeodomain protein fushi tarazu (ftz) is analyzed. We find that specific DNA binding is an important but not sufficient determinant of the functional specificity of ftz in vivo: The ftz DNA-binding specificity mutant ftzQ50K retains partial ftz wild-type activity in gene activation and phenotypic rescue assays. Furthermore, specificity mutations in a ftz-in vivo binding site only partially reduce enhancer activity as compared to null mutations of this site. Despite bicoid-like DNA-binding specificity ftzQ50K does not activate natural or artificial bcd target genes in the realms of ftz. These results are discussed in the light of recent observations on the mechanism of action of the yeast homeodomain protein alpha 2. Images PMID:8434005

  9. Quantitative Analysis of Cellular Senescence in Culture and In Vivo.

    PubMed

    Zhao, Jing; Fuhrmann-Stroissnigg, Heike; Gurkar, Aditi U; Flores, Rafael R; Dorronsoro, Akaitz; Stolz, Donna B; St Croix, Claudette M; Niedernhofer, Laura J; Robbins, Paul D

    2017-01-05

    Cellular senescence refers to the irreversible growth arrest of normally dividing cells in response to various types of stress. Cellular senescence is induced by telomere shortening due to repeated cell division, which causes a DNA damage response, as well as genotoxic, oxidative, and inflammatory stress. Strong mitogenic signaling, such as oncogene activation, also drives cells into a senescent state. Senescent cells express a specific subset of genes, termed the senescence-associated secretory phenotype (SASP), including pro-inflammatory factors, growth factors, and matrix metalloproteinases, which together promote non-cell autonomous, secondary senescence. Clearance of senescent cells that accumulate with age improves health span, implicating cellular senescence as a contributing factor to the aging process. Thus, there is a need for methods to identify and quantify cellular senescence, both in cultured cells and in vivo. Here, methods for the most well-characterized and widely used senescent assays are described, from cell morphology and senescence-associated β-galactosidase (SA-βgal) staining to nuclear biomarkers, SASP, and altered levels of tumor suppressors. © 2017 by John Wiley & Sons, Inc.

  10. Fiberless multicolor neural optoelectrode for in vivo circuit analysis

    NASA Astrophysics Data System (ADS)

    Kampasi, Komal; Stark, Eran; Seymour, John; Na, Kyounghwan; Winful, Herbert G.; Buzsáki, György; Wise, Kensall D.; Yoon, Euisik

    2016-08-01

    Maximizing the potential of optogenetic approaches in deep brain structures of intact animals requires optical manipulation of neurons at high spatial and temporal resolutions, while simultaneously recording electrical data from those neurons. Here, we present the first fiber-less optoelectrode with a monolithically integrated optical waveguide mixer that can deliver multicolor light at a common waveguide port to achieve multicolor modulation of the same neuronal population in vivo. We demonstrate successful device implementation by achieving efficient coupling between a side-emitting injection laser diode (ILD) and a dielectric optical waveguide mixer via a gradient-index (GRIN) lens. The use of GRIN lenses attains several design features, including high optical coupling and thermal isolation between ILDs and waveguides. We validated the packaged devices in the intact brain of anesthetized mice co-expressing Channelrhodopsin-2 and Archaerhodopsin in pyramidal cells in the hippocampal CA1 region, achieving high quality recording, activation and silencing of the exact same neurons in a given local region. This fully-integrated approach demonstrates the spatial precision and scalability needed to enable independent activation and silencing of the same or different groups of neurons in dense brain regions while simultaneously recording from them, thus considerably advancing the capabilities of currently available optogenetic toolsets.

  11. Fiberless multicolor neural optoelectrode for in vivo circuit analysis

    PubMed Central

    Kampasi, Komal; Stark, Eran; Seymour, John; Na, Kyounghwan; Winful, Herbert G.; Buzsáki, György; Wise, Kensall D.; Yoon, Euisik

    2016-01-01

    Maximizing the potential of optogenetic approaches in deep brain structures of intact animals requires optical manipulation of neurons at high spatial and temporal resolutions, while simultaneously recording electrical data from those neurons. Here, we present the first fiber-less optoelectrode with a monolithically integrated optical waveguide mixer that can deliver multicolor light at a common waveguide port to achieve multicolor modulation of the same neuronal population in vivo. We demonstrate successful device implementation by achieving efficient coupling between a side-emitting injection laser diode (ILD) and a dielectric optical waveguide mixer via a gradient-index (GRIN) lens. The use of GRIN lenses attains several design features, including high optical coupling and thermal isolation between ILDs and waveguides. We validated the packaged devices in the intact brain of anesthetized mice co-expressing Channelrhodopsin-2 and Archaerhodopsin in pyramidal cells in the hippocampal CA1 region, achieving high quality recording, activation and silencing of the exact same neurons in a given local region. This fully-integrated approach demonstrates the spatial precision and scalability needed to enable independent activation and silencing of the same or different groups of neurons in dense brain regions while simultaneously recording from them, thus considerably advancing the capabilities of currently available optogenetic toolsets. PMID:27485264

  12. Analysis of an in vivo model to study the interaction of host factors with Candida albicans.

    PubMed Central

    Poor, A H; Cutler, J E

    1981-01-01

    Conditions were established under which membrane diffusion chambers surgically implanted into mice could be used to study interactions between host defense factors and Candida albicans within the chambers. Depending on the size of membrane pores, soluble host substances and phagocytic cells entered the chambers during the first 24 h after chamber implantation. By 7 days in vivo, the membranes of chambers appeared impermeable to these host factors. Mouse phagocytic cells were found to be functional within the in vivo chambers whether the cells emigrated to the chambers on their own accord or were placed there before chamber implantation. Opsonic factors such as antibody and complement appeared to be biologically functional within the in vivo chambers. Conditions suitable for harvesting C. albicans from the chambers also were determined. PMID:7014455

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

    PubMed

    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

  14. Impact of hydrogel nanoparticle size and functionalization on in vivo behavior for lung imaging and therapeutics

    PubMed Central

    Liu, Yongjian; Ibricevic-Richardson, Aida; Cohen, Joel A.; Cohen, Jessica L.; Gunsten, Sean P.; Fréchet, Jean M. J.; Walter, Michael J.; Welch, Michael J.; Brody, Steven L.

    2009-01-01

    Polymer chemistry offers the possibility of synthesizing multifunctional nanoparticles which incorporate moieties that enhance diagnostic and therapeutic targeting of cargo delivery to the lung. However, since rules for predicting particle behavior following modification are not well defined, it is essential that probes for tracking fate in vivo are also included. Accordingly, we designed polyacrylamide-based hydrogel particles of differing sizes, functionalized with a nona-arginine cell-penetrating peptide (Arg9), and labeled with imaging components to assess lung retention and cellular uptake after intratracheal administration. Radiolabeled microparticles (1–5 µm diameter) and nanoparticles (20–40 nm diameter) without and with Arg9 showed diffuse airspace distribution by positron emission tomography imaging. Biodistribution studies revealed that particle clearance and extrapulmonary distribution was, in part, size dependent. Microparticles were rapidly cleared by mucociliary routes but unexpectedly, also through the circulation. In contrast, nanoparticles had prolonged lung retention enhanced by Arg9 and were significantly restricted to the lung. For all particle types, uptake was predominant in alveolar macrophages, and, to a lesser extent, lung epithelial cells. In general, particles did not induce local inflammatory responses, with the exception of microparticles bearing Arg9. Whereas microparticles may be advantageous for short-term applications, nano-sized particles constitute an efficient high-retention and non-inflammatory vehicle for the delivery of diagnostic imaging agents and therapeutics to lung airspaces and alveolar macrophages that can be enhanced by Arg9. Importantly, our results show that minor particle modifications may significantly impact in vivo behavior within the complex environments of the lung, underscoring the need for animal modeling. PMID:19852512

  15. Hippocampal shape analysis in Alzheimer's disease using functional data analysis.

    PubMed

    Epifanio, Irene; Ventura-Campos, Noelia

    2014-02-28

    The hippocampus is one of the first affected regions in Alzheimer's disease. The left hippocampi of control subjects, patients with mild cognitive impairment and patients with Alzheimer's disease are represented by spherical harmonics. Functional data analysis is used in the hippocampal shape analysis. Functional principal component analysis and functional independent component analysis are defined for multivariate functions with two arguments. A functional linear discriminant function is also defined. Comparisons with other approaches are carried out. Our functional approach gives promising results, especially in shape classification.

  16. In vivo studies of silk based gold nano-composite conduits for functional peripheral nerve regeneration.

    PubMed

    Das, Suradip; Sharma, Manav; Saharia, Dhiren; Sarma, Kushal Konwar; Sarma, Monalisa Goswami; Borthakur, Bibhuti Bhusan; Bora, Utpal

    2015-09-01

    We report a novel silk-gold nanocomposite based nerve conduit successfully tested in a neurotmesis grade sciatic nerve injury model in rats over a period of eighteen months. The conduit was fabricated by adsorbing gold nanoparticles onto silk fibres and transforming them into a nanocomposite sheet by electrospinning which is finally given a tubular structure by rolling on a stainless steel mandrel of chosen diameter. The conduits were found to promote adhesion and proliferation of Schwann cells in vitro and did not elicit any toxic or immunogenic responses in vivo. We also report for the first time, the monitoring of muscular regeneration post nerve conduit implantation by recording motor unit potentials (MUPs) through needle electromyogram. Pre-seeding the conduits with Schwann cells enhanced myelination of the regenerated tissue. Histo-morphometric and electrophysiological studies proved that the nanocomposite based conduits pre-seeded with Schwann cells performed best in terms of structural and functional regeneration of severed sciatic nerves. The near normal values of nerve conduction velocity (50 m/sec), compound muscle action potential (29.7 mV) and motor unit potential (133 μV) exhibited by the animals implanted with Schwann cell loaded nerve conduits in the present study are superior to those observed in previous reports with synthetic materials as well as collagen based nerve conduits. Animals in this group were also able to perform complex locomotory activities like stretching and jumping with excellent sciatic function index (SFI) and led a normal life.

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

    NASA Technical Reports Server (NTRS)

    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.

  18. Twins, quadruplexes, and more: functional aspects of native and engineered RNA self-assembly in vivo.

    PubMed

    Lease, Richard A; Arluison, Véronique; Lavelle, Christophe

    2012-03-01

    The primacy and power of RNA in governing many processes of life has begun to be more fully appreciated in both the discovery and inventive sciences. A variety of RNA interactions regulate gene expression, and structural self-assembly underlies many of these processes. The understanding sparked by these discoveries has inspired and informed the engineering of novel RNA structures, control elements, and genetic circuits in cells. Many of these engineered systems are built up fundamentally from RNA-RNA interactions, often combining modular, rational design with functional selection and screening. It is therefore useful to review the particular class of RNA-based regulatory mechanisms that rely on RNA self-assembly either through homomeric (self-self) or heteromeric (self-nonself) RNA-RNA interactions. Structures and sequence elements within individual RNAs create a basis for the pairing interactions, and in some instances can even lead to the formation of RNA polymers. Example systems of dimers, multimers, and polymers are reviewed in this article in the context of natural systems, wherein the function and impact of self-assemblies are understood. Following this, a brief overview is presented of specific engineered RNA self-assembly systems implemented in vivo, with lessons learned from both discovery and engineering approaches to RNA-RNA self-assembly.

  19. Animal Models for Studying the In Vivo Functions of Cell Cycle CDKs.

    PubMed

    Risal, Sanjiv; Adhikari, Deepak; Liu, Kui

    2016-01-01

    Multiple Cdks (Cdk4, Cdk6, and Cdk2) and a mitotic Cdk (Cdk1) are involved in cell cycle progression in mammals. Cyclins, Cdk inhibitors, and phosphorylations (both activating and inhibitory) at different cellular levels tightly modulate the activities of these kinases. Based on the results of biochemical studies, it was long believed that different Cdks functioned at specific stages during cell cycle progression. However, deletion of all three interphase Cdks in mice affected cell cycle entry and progression only in certain specialized cells such as hematopoietic cells, beta cells of the pancreas, pituitary lactotrophs, and cardiomyocytes. These genetic experiments challenged the prevailing biochemical model and established that Cdks function in a cell-specific, but not a stage-specific, manner during cell cycle entry and the progression of mitosis. Recent in vivo studies have further established that Cdk1 is the only Cdk that is both essential and sufficient for driving the resumption of meiosis during mouse oocyte maturation. These genetic studies suggest a minimal-essential cell cycle model in which Cdk1 is the central regulator of cell cycle progression. Cdk1 can compensate for the loss of the interphase Cdks by forming active complexes with A-, B-, E-, and D-type Cyclins in a stepwise manner. Thus, Cdk1 plays an essential role in both mitosis and meiosis in mammals, whereas interphase Cdks are dispensable.

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

    PubMed

    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.

  1. In vivo imaging of cardiac development and function in zebrafish using light sheet microscopy.

    PubMed

    Weber, Michael; Huisken, Jan

    2015-01-01

    Detailed studies of heart development and function are crucial for our understanding of cardiac failures and pave the way for better diagnostics and treatment. However, the constant motion and close incorporation into the cardiovascular system prevent in vivo studies of the living, unperturbed heart. The complementary strengths of the zebrafish model and light sheet microscopy provide a useful platform to fill this gap. High-resolution images of the embryonic vertebrate heart are now recorded from within the living animal: deep inside the unperturbed heart we can follow cardiac contractions and measure action potentials and calcium transients. Three-dimensional reconstructions of the entire beating heart with cellular resolution give new insights into its ever-changing morphology and facilitate studies into how individual cells form the complex cardiac network. In addition, cardiac dynamics and robustness are now examined with targeted optical manipulation. Overall, the combination of zebrafish and light sheet microscopy represents a promising addition for cardiac research and opens the door to a better understanding of heart function and development.

  2. Proteomic Landscape of Tissue-Specific Cyclin E Functions in Vivo

    PubMed Central

    Odajima, Junko; Jung, Piotr; Ndassa-Colday, Yasmine; Ficaro, Scott; Geng, Yan; Marco, Eugenio; Michowski, Wojciech; Wang, Yaoyu E.; DeCaprio, James A.; Litovchick, Larisa; Marto, Jarrod; Sicinski, Piotr

    2016-01-01

    E-type cyclins (cyclins E1 and E2) are components of the cell cycle machinery that has been conserved from yeast to humans. The major function of E-type cyclins is to drive cell division. It is unknown whether in addition to their ‘core’ cell cycle functions, E-type cyclins also perform unique tissue-specific roles. Here, we applied high-throughput mass spectrometric analyses of mouse organs to define the repertoire of cyclin E protein partners in vivo. We found that cyclin E interacts with distinct sets of proteins in different compartments. These cyclin E interactors are highly enriched for phosphorylation targets of cyclin E and its catalytic partner, the cyclin-dependent kinase 2 (Cdk2). Among cyclin E interactors we identified several novel tissue-specific substrates of cyclin E-Cdk2 kinase. In proliferating compartments, cyclin E-Cdk2 phosphorylates Lin proteins within the DREAM complex. In the testes, cyclin E-Cdk2 phosphorylates Mybl1 and Dmrtc2, two meiotic transcription factors that represent key regulators of spermatogenesis. In embryonic and adult brains cyclin E interacts with proteins involved in neurogenesis, while in adult brains also with proteins regulating microtubule-based processes and microtubule cytoskeleton. We also used quantitative proteomics to demonstrate re-wiring of the cyclin E interactome upon ablation of Cdk2. This approach can be used to study how protein interactome changes during development or in any pathological state such as aging or cancer. PMID:27828963

  3. Polyglycerolsulfate Functionalized Gold Nanorods as Optoacoustic Signal Nanoamplifiers for In Vivo Bioimaging of Rheumatoid Arthritis

    PubMed Central

    Vonnemann, Jonathan; Beziere, Nicolas; Böttcher, Christoph; Riese, Sebastian B.; Kuehne, Christian; Dernedde, Jens; Licha, Kai; von Schacky, Claudio; Kosanke, Yvonne; Kimm, Melanie; Meier, Reinhard; Ntziachristos, Vasilis; Haag, Rainer

    2014-01-01

    We have synthesized a targeted imaging agent for rheumatoid arthritis based on polysulfated gold nanorods. The CTAB layer on gold nanorods was first replaced with PEG-thiol and then with dendritic polyglycerolsulfate at elevated temperature, which resulted in significantly reduced cytotoxicity compared to polyanionic gold nanorods functionalized by non-covalent approaches. In addition to classical characterization methods, we have established a facile UV-VIS based BaCl2 agglomeration assay to confirm a quantitative removal of unbound ligand. With the help of a competitive surface plasmon resonance-based L-selectin binding assay and a leukocyte adhesion-based flow cell assay, we have demonstrated the high inflammation targeting potential of the synthesized gold nanorods in vitro. In combination with the surface plasmon resonance band of AuNRs at 780 nm, these findings permitted the imaging of inflammation in an in vivo mouse model for rheumatoid arthritis with high contrast using multispectral optoacoustic tomography. The study offers a robust method for otherwise difficult to obtain covalently functionalized polyanionic gold nanorods, which are suitable for biological applications as well as a low-cost, actively targeted, and high contrast imaging agent for the diagnosis of rheumatoid arthritis. This paves the way for further research in other inflammation associated pathologies, in particular, when photothermal therapy can be applied. PMID:24723984

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

    PubMed

    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.

  5. Classical and adaptive control of ex vivo skeletal muscle contractions using Functional Electrical Stimulation (FES)

    PubMed Central

    Shoemaker, Adam; Grange, Robert W.; Abaid, Nicole; Leonessa, Alexander

    2017-01-01

    Functional Electrical Stimulation is a promising approach to treat patients by stimulating the peripheral nerves and their corresponding motor neurons using electrical current. This technique helps maintain muscle mass and promote blood flow in the absence of a functioning nervous system. The goal of this work is to control muscle contractions from FES via three different algorithms and assess the most appropriate controller providing effective stimulation of the muscle. An open-loop system and a closed-loop system with three types of model-free feedback controllers were assessed for tracking control of skeletal muscle contractions: a Proportional-Integral (PI) controller, a Model Reference Adaptive Control algorithm, and an Adaptive Augmented PI system. Furthermore, a mathematical model of a muscle-mass-spring system was implemented in simulation to test the open-loop case and closed-loop controllers. These simulations were carried out and then validated through experiments ex vivo. The experiments included muscle contractions following four distinct trajectories: a step, sine, ramp, and square wave. Overall, the closed-loop controllers followed the stimulation trajectories set for all the simulated and tested muscles. When comparing the experimental outcomes of each controller, we concluded that the Adaptive Augmented PI algorithm provided the best closed-loop performance for speed of convergence and disturbance rejection. PMID:28273101

  6. Immune Signature of Enhanced Functional Avidity CD8+ T Cells in vivo Induced by Vaccinia Vectored Vaccine

    PubMed Central

    Hu, Zhidong; Zhu, Lingyan; Wang, Jing; Wan, Yanmin; Yuan, Songhua; Chen, Jian; Ding, Xiangqing; Qiu, Chenli; Zhang, Xiaoyan; Qiu, Chao; Xu, Jianqing

    2017-01-01

    Functional avidity of T cells is a critical determinant for clearing viral infection and eliminating tumor. Understanding how functional avidity is maintained in T cells is imperative for immunotherapy. However, studies systematically characterize T cell with high functional avidity induced in vivo are still lacking. Previously, we and others found vaccinia vectored vaccine (VACV) induced antigen-specific CD8+ T cells with relatively high functional avidity to those from DNA vaccine. Herein, we used functional, immune phenotyping and transcriptomic studies to define the immune signature of these CD8+ T cells with high functional avidity. Antigen-specific CD8+ T cells induced by VACV executed superior in vivo killing activity and displayed a distinct transcriptional profile, whereas no significantly differences were found in composition of memory sub-populations and cytokine poly-functionality. Transcriptional analyses revealed unique features of VACV induced CD8+ T cells in several biological processes, including transport, cell cycle, cell communication and metabolic processes. In summary, we characterize CD8+ T cells of high functional avidity induced in vivo by VACV, which not only improves our understanding of adaptive T cell immunity in VACV vaccination, but also provides clues to modulate functional avidity of CD8+ T cells for T cell based immunotherapy. PMID:28155878

  7. In vivo analysis of human nucleoporin repeat domain interactions

    PubMed Central

    Xu, Songli; Powers, Maureen A.

    2013-01-01

    The nuclear pore complex (NPC), assembled from ∼30 proteins termed nucleoporins (Nups), mediates selective nucleocytoplasmic trafficking. A subset of nucleoporins bear a domain with multiple phenylalanine–glycine (FG) motifs. As binding sites for transport receptors, FG Nups are critical in translocation through the NPC. Certain FG Nups are believed to associate via low-affinity, cohesive interactions to form the permeability barrier of the pore, although the form and composition of this functional barrier are debated. We used green fluorescent protein–Nup98/HoxA9 constructs with various numbers of repeats and also substituted FG domains from other nucleoporins for the Nup98 domain to directly compare cohesive interactions in live cells by fluorescence recovery after photobleaching (FRAP). We find that cohesion is a function of both number and type of FG repeats. Glycine–leucine–FG (GLFG) repeat domains are the most cohesive. FG domains from several human nucleoporins showed no interactions in this assay; however, Nup214, with numerous VFG motifs, displayed measurable cohesion by FRAP. The cohesive nature of a human nucleoporin did not necessarily correlate with that of its yeast orthologue. The Nup98 GLFG domain also functions in pore targeting through binding to Nup93, positioning the GLFG domain in the center of the NPC and supporting a role for this nucleoporin in the permeability barrier. PMID:23427268

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

    PubMed

    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.

  9. Cardiac magnetic resonance, transthoracic and transoesophageal echocardiography: a comparison of in vivo assessment of ventricular function in rats.

    PubMed

    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.

  10. In vivo expression and function of hybrid Ia dimers (E alpha A beta) in recombinant and transgenic mice

    PubMed Central

    1989-01-01

    We have found cell surface expression of an E alpha molecule in recombinant and transgenic mouse strains lacking an E beta molecule. Flow cytometry has shown low level expression of E alpha in B10.RQB3 (I- AqEk alpha) and B10.RFB2 (I-AfEk alpha) mice. We have also found that B10.Q (H-2q) mice can express the Ek alpha transgene. Since these strains do not have functional E beta chains, we propose that the E alpha A beta hybrid dimers are formed in low numbers and can be picked up by FACS analysis. So far we have not been able to identify these hybrid molecules by cytotoxicity or immunoprecipitation. The E alpha/A beta molecule can function in vivo during thymic selection in the clonal deletion of two V beta TCR subsets, V beta 11 and V beta 6, which have been shown to interact with the intact I-E molecule. PMID:2788700

  11. A novel method for determining human ex vivo submaximal skeletal muscle mitochondrial function

    PubMed Central

    Hey-Mogensen, Martin; Gram, Martin; Jensen, Martin Borch; Lund, Michael Taulo; Hansen, Christina Neigaard; Scheibye-Knudsen, Morten; Bohr, Vilhelm A; Dela, Flemming

    2015-01-01

    Abstract Despite numerous studies, there is no consensus about whether mitochondrial function is altered with increased age. The novelty of the present study is the determination of mitochondrial function at submaximal activity rates, which is more physiologically relevant than the ex vivo functionality protocols used previously. Muscle biopsies were taken from 64 old or young male subjects (aged 60–70 or 20–30 years). Aged subjects were recruited as trained or untrained. Muscle biopsies were used for the isolation of mitochondria and subsequent measurements of DNA repair, anti-oxidant capacity and mitochondrial protein levels (complexes I–V). Mitochondrial function was determined by simultaneous measurement of oxygen consumption, membrane potential and hydrogen peroxide emission using pyruvate + malate (PM) or succinate + rotenone (SR) as substrates. Proton leak was lower in aged subjects when determined at the same membrane potential and was unaffected by training status. State 3 respiration was lower in aged untrained subjects. This effect, however, was alleviated in aged trained subjects. H2O2 emission with PM was higher in aged subjects, and was exacerbated by training, although it was not changed when using SR. However, with a higher manganese superoxide dismuthase content, the trained aged subjects may actually have lower or similar mitochondrial superoxide emission compared to the untrained subjects. We conclude that ageing and the physical activity level in aged subjects are both related to changes in the intrinsic functionality of the mitochondrion in skeletal muscle. Both of these changes could be important factors in determining the metabolic health of the aged skeletal muscle cell. Key points The present study utilized a novel method aiming to investigate mitochondrial function in human skeletal muscle at submaximal levels and at a predefined membrane potential. The effect of age and training status was investigated using a cross

  12. Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo.

    PubMed

    Najm, Fadi J; Madhavan, Mayur; Zaremba, Anita; Shick, Elizabeth; Karl, Robert T; Factor, Daniel C; Miller, Tyler E; Nevin, Zachary S; Kantor, Christopher; Sargent, Alex; Quick, Kevin L; Schlatzer, Daniela M; Tang, Hong; Papoian, Ruben; Brimacombe, Kyle R; Shen, Min; Boxer, Matthew B; Jadhav, Ajit; Robinson, Andrew P; Podojil, Joseph R; Miller, Stephen D; Miller, Robert H; Tesar, Paul J

    2015-06-11

    Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor

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

    PubMed

    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. Human whole-blood culture system for ex vivo characterization of designer-cell function.

    PubMed

    Schukur, Lina; Geering, Barbara; Fussenegger, Martin

    2016-03-01

    Encapsulated designer cells implanted into mice are currently used to validate the efficacy of therapeutic gene networks for the diagnosis and treatment of various human diseases in preclinical research. Because many human conditions cannot be adequately replicated by animal models, complementary and alternative procedures to test future treatment strategies are required. Here we describe a novel approach utilizing an ex vivo human whole-blood culture system to validate synthetic biology-inspired designer cell-based treatment strategies. The viability and functionality of transgenic mammalian designer cells co-cultured with primary human immune cells were characterized. We demonstrated that transgenic mammalian designer cells required adequate insulation from the human blood microenvironment to maintain viability and functionality. The biomaterial alginate-(poly-l-lysine)-alginate used to encapsulate the transgenic designer cells did neither affect the viability of primary granulocytes and lymphocytes nor the functionality of lymphocytes. Additionally, alginate-encapsulated transgenic designer cells remained responsive to the release of the pro-inflammatory cytokine tumor necrosis factor (TNF) from the whole-blood culture upon exposure to bacterial lipopolysaccharide (LPS). TNF diffused into the alginate capsules, bound to the specific TNF receptors on the transgenic designer cells' surface and triggered the expression of the reporter gene SEAP (human placental secreted alkaline phosphatase) that was rewired to the TNF-specific signaling cascade. Human whole-blood culture systems can therefore be considered as valuable complementary assays to animal models for the validation of synthetic circuits in genetically modified mammalian cells and may speed up preclinical research in a world of personalized medicine.

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

    PubMed

    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.

  16. Recovery of macular pigment spectrum in vivo using hyperspectral image analysis

    NASA Astrophysics Data System (ADS)

    Fawzi, Amani A.; Lee, Noah; Acton, Jennifer H.; Laine, Andrew F.; Smith, R. Theodore

    2011-10-01

    We investigated the feasibility of a novel method for hyperspectral mapping of macular pigment (MP) in vivo. Six healthy subjects were recruited for noninvasive imaging using a snapshot hyperspectral system. The three-dimensional full spatial-spectral data cube was analyzed using non-negative matrix factorization (NMF), wherein the data was decomposed to give spectral signatures and spatial distribution, in search for the MP absorbance spectrum. The NMF was initialized with the in vitro MP spectrum and rank 4 spectral signature decomposition was used to recover the MP spectrum and optical density in vivo. The recovered MP spectra showed two peaks in the blue spectrum, characteristic of MP, giving a detailed in vivo demonstration of these absorbance peaks. The peak MP optical densities ranged from 0.08 to 0.22 (mean 0.15+/-0.05) and became spatially negligible at diameters 1100 to 1760 μm (4 to 6 deg) in the normal subjects. This objective method was able to exploit prior knowledge (the in vitro MP spectrum) in order to extract an accurate in vivo spectral analysis and full MP spatial profile, while separating the MP spectra from other ocular absorbers. Snapshot hyperspectral imaging in combination with advanced mathematical analysis provides a simple cost-effective approach for MP mapping in vivo.

  17. Structure and function analysis of protein-nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein-nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  18. Function of dopamine transporter is compromised in DYT1 transgenic animal model in vivo

    PubMed Central

    Hewett, Jeff; Johansen, Peter; Sharma, Nutan; Standaert, David; Balcioglu, Aygul

    2011-01-01

    Early onset torsion dystonia (DYT1), the most common form of hereditary primary dystonia, is caused by a mutation in the TOR1A gene, which codes for the protein, torsinA. We previously examined the effect of the human mutant torsinA on striatal dopaminergic function in a conventional transgenic mouse model of DYT1 dystonia (hMT1), in which human mutant torsinA is expressed under the cytomegalovirus promotor. Systemic administration of amphetamine did not increase dopamine (DA) release as efficiently in these mice as compared with wild-type transgenic and non-transgenic mice. We, now, studied the contribution of the DA transporter (DAT) to amphetamine-induced DA release in hMT1 transgenic mice using in vivo no-net flux microdialysis. This method applies different concentrations of DA through the microdialysis probe and measures DA concentration at the output of the probe following an equilibrium period. The slope (extraction fraction) is the measure of the DAT activity in vivo. The slope for hMT1 transgenic mice was 0.58 ± 0.07 and for non-transgenic animals, 0.87 ± 0.06 (p < 0.05). We further investigated the efficacy of nomifensine (a specific DAT inhibitor) in inhibiting amphetamine-induced DA release. Local application of nomifensine 80 min before the systemic application of amphetamine inhibited DA release in both transgenic mice and their non-transgenic littermates. The efficiency of the inhibition appeared to be different, with mean values of 48% for hMT1 transgenic mice versus 84% for non-transgenic littermates. Moreover, we have evaluated basal and amphetamine-induced locomotion in hMT1 transgenic mice compared with their non-transgenic littermates, using an O-maze behavioral chamber. Basal levels of locomotion in the hMT1 transgenic mice showed that they moved much less than their non-transgenic littermates (0.9 ± 0.3 m for transgenic mice vs. 2.4 ± 0.7 m for non-transgenic littermates, p < 0.05). This relative reduction in locomotion was also observed

  19. Development of Spectral Domain Optical Coherence Tomography for in vivo Functional Imaging of Biological Tissues

    NASA Astrophysics Data System (ADS)

    An, Lin

    Optical coherence tomography is a rapidly developing optical imaging modality capable of noninvasively providing depth resolved information of biological tissue at micrometer scale. In this thesis, we described several OCT technologies that can be used to double the imaging depth, realize functional vasculature imaging of biological tissue and increase the imaging speed of OCT system. Aim 1: Use of a scanner to introduce spatial frequency modulation to OCT spectral interferograms for in vivo full-range Fourier-domain optical coherence tomography. A novel method was developed that could easily introduce a modulation frequency onto the X-direction (i.e., B-scan) of the FDOCT scanning system, enabling full-range Fourier-domain Optical Coherence Tomography (frFDOCT). Compared to the conventional FDOCT system, the newly developed frFDOCT system can provide increased system sensitivity and deeper imaging depth. The previous technology that can achieve frFDOCT either needed multiple steps for data capturing, which is time consuming, or required additional components which increased the system's complexity. The newly developed method generates a modulation spatial frequency in the spectral interferogram by simply offsetting the probe beam at the X-scanner. Aim 2: Using optical micro-angiography to achieve in vivo volumetric imaging of vascular perfusion within human retina and choroids. Optical Micro-Angiography (OMAG) is a functional extension of FDOCT technology. It can achieve visualization of vasculature network of biological tissue. In order to apply the OMAG method to image vasculature map of human retina and choroid, a phase compensation algorithm was developed, which could minimize the motion artifacts generated by the movements of human eye and head. Aim 3: Developing ultrahigh sensitive optical micro-angiography to achieve micro vasculature imaging of biological tissue. To improve the vasculature image quality, we developed ultrahigh sensitive OMAG (UHS

  20. Randomized Controlled Trial of "Mind Reading" and In Vivo Rehearsal for High-Functioning Children with ASD

    ERIC Educational Resources Information Center

    Thomeer, Marcus L.; Smith, Rachael A.; Lopata, Christopher; Volker, Martin A.; Lipinski, Alanna M.; Rodgers, Jonathan D.; McDonald, Christin A.; Lee, Gloria K.

    2015-01-01

    This randomized controlled trial evaluated the efficacy of a computer software (i.e., "Mind Reading") and in vivo rehearsal treatment on the emotion decoding and encoding skills, autism symptoms, and social skills of 43 children, ages 7-12 years with high-functioning autism spectrum disorder (HFASD). Children in treatment (n = 22)…

  1. ANALYSIS OF IN VITRO AND IN VIVO DNA STRAND BREAKS INDUCED BY TRIHALOMETHANES (THMS)

    EPA Science Inventory

    Analysis of In Vitro and In Vivo DNA Strand Breaks Induced by Trihalomethanes (TRMs)

    The THMs are the most widely distributed and the most concentrated of the cWorine disinfection by-products (D BPs) found in finished drinking water. All of the THMs, cWoroform (CHCI3), br...

  2. 40 CFR 798.5385 - In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 31 2010-07-01 2010-07-01 true In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis. 798.5385 Section 798.5385 Protection of Environment ENVIRONMENTAL... used. Examples of commonly used rodent species are rats, mice, and hamsters. (ii) Age. Healthy...

  3. Functional Anatomy of the Thalamus as a Model of Integrated Structural and Functional Connectivity of the Human Brain In Vivo.

    PubMed

    Mastropasqua, Chiara; Bozzali, Marco; Spanò, Barbara; Koch, Giacomo; Cercignani, Mara

    2015-07-01

    While methods of measuring non-invasively both, functional and structural brain connectivity are available, the degree of overlap between them is still unknown. In this paper this issue is addressed by investigating the connectivity pattern of a brain structure with many, well characterized structural connections, namely the thalamus. Diffusion-weighted and resting state (RS) functional MRI (fMRI) data were collected in a group of 38 healthy participants. Probabilistic tractography was performed to parcellate the thalamus into regions structurally connected to different cortical areas. The resulting regions were used as seeds for seed-based analysis of RS fMRI data. The tractographic parcellation was thus cross-validated against functional connectivity data by evaluating the overlap between the functional and structural thalamo-cortical connections originating from the parcellated regions. Our data show only a partial overall correspondence between structural and functional connections, in the same group of healthy individuals, thus suggesting that the two approaches provide complementary and not overlapping information. Future studies are warranted to extend the results we obtained in the thalamus to other structures, and to confirm that the mechanisms behind functional connectivity are more complex than just expressing structural connectivity.

  4. Teacher Praise: A Functional Analysis.

    ERIC Educational Resources Information Center

    Brophy, Jere

    1981-01-01

    Teacher praise typically does not function as a reinforcer. Rather, it is reactive to and under the control of student behavior. Its effects must be understood using concepts from attribution and social learning/reinforcement theories. (Author/GK)

  5. Functional Techniques for Data Analysis

    NASA Technical Reports Server (NTRS)

    Tomlinson, John R.

    1997-01-01

    This dissertation develops a new general method of solving Prony's problem. Two special cases of this new method have been developed previously. They are the Matrix Pencil and the Osculatory Interpolation. The dissertation shows that they are instances of a more general solution type which allows a wide ranging class of linear functional to be used in the solution of the problem. This class provides a continuum of functionals which provide new methods that can be used to solve Prony's problem.

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

    PubMed

    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.

  7. Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo

    PubMed Central

    2016-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79–80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type–specific disorders that often transition from hypoproliferative to hyperproliferative growth. PMID:25706898

  8. In vivo effects of Eurycoma longifolia Jack (Tongkat Ali) extract on reproductive functions in the rat.

    PubMed

    Solomon, M C; Erasmus, N; Henkel, R R

    2014-05-01

    An aqueous extract of Eurycoma longifolia (Tongkat Ali; TA) roots is traditionally used to enhance male sexuality. Because previous studies are limited to only few sperm parameters or testosterone concentration, this study investigated the in vivo effects of TA on body and organ weight as well as functional sperm parameters in terms of safety and efficacy in the management of male infertility. Forty-two male rats were divided into a control, low-dose (200 mg kg(-1) BW) and high-dose (800 mg kg(-1) BW) group (n = 14). Rats were force-fed for 14 days and then sacrificed. Total body and organ weights of the prostate, testes, epididymides, gastrocnemius muscle and the omentum were recorded. Moreover, testosterone concentration, sperm concentration, motility, velocity, vitality, acrosome reaction and mitochondrial membrane potential (MMP) were assessed. Whilst TA decreased BW by 5.7% (P = 0.0276) and omentum fat by 31.9% (P = 0.0496), no changes in organ weights were found for the prostate, testes and epididymides. Testosterone concentration increased by 30.2% (P = 0.0544). Muscle weight also increased, yet not significantly. Whilst sperm concentration, total and progressive motility and vitality increased significantly, MMP improved markedly (P = 0.0765) by 25.1%. Because no detrimental effect could be observed, TA appears safe for possible treatment of male infertility and ageing male problems.

  9. In vivo functional and myeloarchitectonic mapping of human primary auditory areas

    PubMed Central

    Dick, Frederic; Tierney, Adam Taylor; Lutti, Antoine; Josephs, Oliver; Sereno, Martin I.; Weiskopf, Nikolaus

    2012-01-01

    In contrast to vision, where retinotopic mapping alone can define areal borders, primary auditory areas such as A1 are best delineated by combining in vivo tonotopic mapping with post mortem cyto- or myelo-architectonics from the same individual. We combined high-resolution (800 μm) quantitative T1 mapping with phase-encoded tonotopic methods to map primary auditory areas (A1 and R) within the ‘auditory core’ of human volunteers. We first quantitatively characterize the highly myelinated auditory core in terms of shape, area, cortical depth profile, and position, with our data showing considerable correspondence to post-mortem myeloarchitectonic studies, both in cross-participant averages and in individuals. The core region contains two ‘mirror-image‘ tonotopic maps oriented along the same axis as observed in macaque and owl monkey. We suggest that thee two maps within the core are the human analogues of primate auditory areas A1 and R. The core occupies a much smaller portion of tonotopically organized cortex on the superior temporal plane and gyrus than is generally supposed. The multi-modal approach to defining the auditory core will facilitate investigations of structure-function relationships, comparative neuroanatomical studies, and promises new biomarkers for diagnosis and clinical studies. PMID:23152594

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

    PubMed Central

    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

  11. Functional integrity of the interrenal tissue of yellow perch from contaminated sites tested in vivo

    SciTech Connect

    Girard, C.; Brodeur, J.C.; Hontela, A.

    1995-12-31

    The normal activation of the hypothalamo-pituitary-interrenal axis (HPI axis) in response to capture is disrupted in fish subjected to life-long exposure to heavy metals, PCBs and PAHs. The ability to increase plasma cortisol in yellow perch (Perca flavescens) from sites contaminated by heavy metals and organic compounds, and from a reference site was assessed by the Capture stress test and by the ACTH Challenge test, a new standardized in vivo method designed for field studies. The effects of seasonal factors, such as temperature and gonadal maturity on these tests were investigated. Measures of liver and muscle glycogen and histopathology were made to further characterize the biochemical and structural changes that may occur along with hormonal changes. The Capture stress test showed that an acute source of stress induced a lower cortisol response in fish from the highly contaminated site compared to the reference site, revealing a functional impairment of the HPI axis. The ACTH Challenge test showed that the hormonal responsiveness of the cortisol-secreting interrenal tissue, stimulated by a standard dose of ACTH injected i.p., was lower in fish from the highly contaminated site than the reference site. Spring is the season during which the impairment was the most evident. The possibility of using the reduced capacity of feral fish to respond to a standardized ACTH Challenge as an early bioindicator of toxic stress is discussed.

  12. The functional response of upstream DNA to dynamic supercoiling in vivo.

    PubMed

    Kouzine, Fedor; Sanford, Suzanne; Elisha-Feil, Zichrini; Levens, David

    2008-02-01

    Because RNA polymerase is a powerful motor, transmission of transcription-generated forces might directly alter DNA structure, chromatin or gene activity in mammalian cells. Here we show that transcription-generated supercoils streaming dynamically from active promoters have considerable consequences for DNA structure and function in cells. Using a tamoxifen-activatable Cre recombinase to excise a test segment of chromatin positioned between divergently transcribed metallothionein-IIa promoters, we found the degree of dynamic supercoiling to increase as transcription intensified, and it was very sensitive to the specific arrangement of promoters and cis elements. Using psoralen as an in vivo probe confirmed that, during transcription, sufficient supercoiling is produced to enable transitions to conformations other than B-DNA in elements such as the human MYC far upstream element (FUSE), which in turn recruit structure-sensitive regulatory proteins, such as FUSE Binding Protein (FBP) and FBP-Interacting Repressor (FIR). These results indicate that mechanical stresses, constrained by architectural features of DNA and chromatin, may broadly contribute to gene regulation.

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

    PubMed

    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.

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

    PubMed

    Burgess, Rebecca C; Lisby, Michael; Altmannova, Veronika; Krejci, Lumir; Sung, Patrick; Rothstein, Rodney

    2009-06-15

    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 removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR.

  15. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo.

    PubMed

    de la Cruz, Jesús; Karbstein, Katrin; Woolford, John L

    2015-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79-80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type-specific disorders that often transition from hypoproliferative to hyperproliferative growth.

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

    PubMed

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

    2017-03-06

    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.

  17. In vivo and in vitro effects of 1,1-dimethylhydrazine on selected immune functions.

    PubMed

    Tarr, M J; Olsen, R G; Jacobs, D L

    1982-04-01

    The in vivo phase of the experiments reported here include the evaluation of immune function after short-or long-term treatment of mice with 1,1-dimethylhydrazine (UDMH). Long-term exposure (3 injections/week for 14 weeks) resulted in increased numbers of Jerne plaque-forming cells, a trend toward decreased induction of suppressor cell activity by concanavalin A (Con A), and no effects on mitogen-induced lymphocyte blast transformation (LBT), compared to saline-treated control mice. These effects were greatest at doses of 10 or 50 mg/kg, while higher doses had less of an effect. In vitro experiments were performed by adding UDMH to normal murine splenocytes in the LBT assay and con A-induced suppressor cell assay. The UDMH induced a significant enhanced response to lipopolysaccharide (LPS) at 10 and 50 micrograms/ml, and a suppressed response to both Con A and LPS at higher concentrations. The UDMH also caused a decrease in suppressor cell activity at 25 micrograms/ml. Selective abrogation of suppressor activity or alteration of the suppressor cell-helper ratio were suggested as possible mechanisms for the enhancement effect associated with UDMH.

  18. Effect of topically applied dexpanthenol on epidermal barrier function and stratum corneum hydration. Results of a human in vivo study.

    PubMed

    Gehring, W; Gloor, M

    2000-07-01

    In a randomized, double-blind, placebo-controlled study the effect of topical dexpanthenol (CAS 81-13-0) formulated in two different lipophilic vehicles on epidermal barrier function in vivo was carried out. Seven days' treatment with dexpanthenol improved stratum corneum hydration and reduced transepidermal water loss. Active treatment was statistically different from the vehicle control on both measures. Our results suggest that topical dexpanthenol formulated in either lipophilic vehicle stabilizes the skin barrier function.

  19. Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies.

    PubMed

    Mayevsky, Avraham; Rogatsky, Gennady G

    2007-02-01

    Normal mitochondrial function is a critical factor in maintaining cellular homeostasis in various organs of the body. Due to the involvement of mitochondrial dysfunction in many pathological states, the real-time in vivo monitoring of the mitochondrial metabolic state is crucially important. This type of monitoring in animal models as well as in patients provides real-time data that can help interpret experimental results or optimize patient treatment. The goals of the present review are the following: 1) to provide an historical overview of NADH fluorescence monitoring and its physiological significance; 2) to present the solid scientific ground underlying NADH fluorescence measurements based on published materials; 3) to provide the reader with basic information on the methodologies used in the past and the current state of the art fluorometers; and 4) to clarify the various factors affecting monitored signals, including artifacts. The large numbers of publications by different groups testify to the valuable information gathered in various experimental conditions. The monitoring of NADH levels in the tissue provides the most important information on the metabolic state of the mitochondria in terms of energy production and intracellular oxygen levels. Although NADH signals are not calibrated in absolute units, their trend monitoring is important for the interpretation of physiological or pathological situations. To understand tissue function better, the multiparametric approach has been developed where NADH serves as the key parameter. The development of new light sources in UV and visible spectra has led to the development of small compact units applicable in clinical conditions for better diagnosis of patients.

  20. Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo

    PubMed Central

    Tietgens, Amber J.; Van Itallie, Christina M.; Vitale‐Cross, Lynn; Jarnik, Michal; Harding, Olivia J.; Anderson, James M.; Gutkind, J. Silvio; Weigert, Roberto; Arias, Irwin M.

    2016-01-01

    Liver kinase B1 (LKB1) and its downstream effector AMP‐activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich‐cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver‐specific (albumin‐Cre) LKB1 knockout mice (LKB1−/−) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1−/− livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6‐carboxyfluorescein diacetate (6‐CFDA), which is processed in hepatocytes into its fluorescent derivative 6‐carboxyfluorescein (6‐CF) and secreted into the canaliculi. In LKB1−/− mice, 6‐CF remained largely in hepatocytes, canalicular secretion was delayed, and 6‐CF appeared in the blood. To test whether 6‐CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6‐CFDA. In wild‐type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1−/− mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. Conclusion: LKB1 plays a critical role in regulating the maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway result in cholestasis. (Hepatology 2016;64:1317‐1329) PMID:27396550

  1. Exposure to low mercury concentration in vivo impairs myocardial contractile function

    SciTech Connect

    Furieri, Lorena Barros; Fioresi, Mirian; Junior, Rogerio Faustino Ribeiro; Bartolome, Maria Visitacion; Fernandes, Aurelia Araujo; Cachofeiro, Victoria; Lahera, Vicente; Salaices, Mercedes; Stefanon, Ivanita; Vassallo, Dalton Valentim

    2011-09-01

    Increased cardiovascular risk after mercury exposure has been described but cardiac effects resulting from controlled chronic treatment are not yet well explored. We analyzed the effects of chronic exposure to low mercury concentrations on hemodynamic and ventricular function of isolated hearts. Wistar rats were treated with HgCl{sub 2} (1st dose 4.6 {mu}g/kg, subsequent dose 0.07 {mu}g/kg/day, im, 30 days) or vehicle. Mercury treatment did not affect blood pressure (BP) nor produced cardiac hypertrophy or changes of myocyte morphometry and collagen content. This treatment: 1) in vivo increased left ventricle end diastolic pressure (LVEDP) without changing left ventricular systolic pressure (LVSP) and heart rate; 2) in isolated hearts reduced LV isovolumic systolic pressure and time derivatives, and {beta}-adrenergic response; 3) increased myosin ATPase activity; 4) reduced Na{sup +}-K{sup +} ATPase (NKA) activity; 5) reduced protein expression of SERCA and phosphorylated phospholamban on serine 16 while phospholamban expression increased; as a consequence SERCA/phospholamban ratio reduced; 6) reduced sodium/calcium exchanger (NCX) protein expression and {alpha}-1 isoform of NKA, whereas {alpha}-2 isoform of NKA did not change. Chronic exposure for 30 days to low concentrations of mercury does not change BP, heart rate or LVSP but produces small but significant increase of LVEDP. However, in isolated hearts mercury treatment promoted contractility dysfunction as a result of the decreased NKA activity, reduction of NCX and SERCA and increased PLB protein expression. These findings offer further evidence that mercury chronic exposure, even at small concentrations, is an environmental risk factor affecting heart function. - Highlights: > Unchanges blood pressure, heart rate, systolic pressure. > Increases end diastolic pressure. > Promotes cardiac contractility dysfunction. > Decreases NKA activity, NCX and SERCA, increases PLB protein expression. > Small

  2. High-resolution imaging and computational analysis of haematopoietic cell dynamics in vivo

    PubMed Central

    Koechlein, Claire S.; Harris, Jeffrey R.; Lee, Timothy K.; Weeks, Joi; Fox, Raymond G.; Zimdahl, Bryan; Ito, Takahiro; Blevins, Allen; Jung, Seung-Hye; Chute, John P.; Chourasia, Amit; Covert, Markus W.; Reya, Tannishtha

    2016-01-01

    Although we know a great deal about the phenotype and function of haematopoietic stem/progenitor cells, a major challenge has been mapping their dynamic behaviour within living systems. Here we describe a strategy to image cells in vivo with high spatial and temporal resolution, and quantify their interactions using a high-throughput computational approach. Using these tools, and a new Msi2 reporter model, we show that haematopoietic stem/progenitor cells display preferential spatial affinity for contacting the vascular niche, and a temporal affinity for making stable associations with these cells. These preferences are markedly diminished as cells mature, suggesting that programs that control differentiation state are key determinants of spatiotemporal behaviour, and thus dictate the signals a cell receives from specific microenvironmental domains. These collectively demonstrate that high-resolution imaging coupled with computational analysis can provide new biological insight, and may in the long term enable creation of a dynamic atlas of cells within their native microenvironment. PMID:27425143

  3. The effect of in vivo endotoxin on myocardial function in vitro.

    PubMed

    Romanosky, A J; Giaimo, M E; Shepherd, R E; Burns, A H

    1986-01-01

    Isolated perfused working hearts from male adult Sprague Dawley rats were used to examine myocardial performance following acute in vivo endotoxin administration (LD50-6-hour). Three hours after endotoxin administration, cardiac output and peak systolic pressure in the isolated perfused working heart were depressed 25-50% over a range of left atrial filling pressures (preload) from 10 to 30 cm of water. Linear regression analysis of the relationship between myocardial work indices, oxygen uptake, and glucose oxidation indicated that the hearts from the endotoxin-treated animals required more oxygen and glucose to perform the same amount of work. Levels of cyclic 3'5' adenosine monophosphate were 72% higher in ventricular tissue from the endotoxin-treated group compared to the hearts of controls. Isoproterenol (10(-9) mol/min) raised levels of the nucleotide to the same final concentration in both groups of hearts, whereas myocardial pressure work in hearts from endotoxin-treated rats was only 70% that of control. Provision of isoproterenol, while increasing mechanical work by the hearts in the endotoxin-treated group, did not induce an increase to the same performance levels as that of control hearts not treated with isoproterenol. These data are consistent with the concept that endotoxemia produces an intrinsic defect in myocardial mechanical performance.

  4. Efficient inhibition of miR-155 function in vivo by peptide nucleic acids

    PubMed Central

    Fabani, Martin M.; Abreu-Goodger, Cei; Williams, Donna; Lyons, Paul A.; Torres, Adrian G.; Smith, Kenneth G. C.; Enright, Anton J.; Gait, Michael J.; Vigorito, Elena

    2010-01-01

    MicroRNAs (miRNAs) play an important role in diverse physiological processes and are potential therapeutic agents. Synthetic oligonucleotides (ONs) of different chemistries have proven successful for blocking miRNA expression. However, their specificity and efficiency have not been fully evaluated. Here, we show that peptide nucleic acids (PNAs) efficiently block a key inducible miRNA expressed in the haematopoietic system, miR-155, in cultured B cells as well as in mice. Remarkably, miR-155 inhibition by PNA in primary B cells was achieved in the absence of any transfection agent. In mice, the high efficiency of the treatment was demonstrated by a strong overlap in global gene expression between B cells isolated from anti-miR-155 PNA-treated and miR-155-deficient mice. Interestingly, PNA also induced additional changes in gene expression. Our analysis provides a useful platform to aid the design of efficient and specific anti-miRNA ONs for in vivo use. PMID:20223773

  5. Ex vivo brain tumor analysis using spectroscopic optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Lenz, Marcel; Krug, Robin; Welp, Hubert; Schmieder, Kirsten; Hofmann, Martin R.

    2016-03-01

    A big challenge during neurosurgeries is to distinguish between healthy tissue and cancerous tissue, but currently a suitable non-invasive real time imaging modality is not available. Optical Coherence Tomography (OCT) is a potential technique for such a modality. OCT has a penetration depth of 1-2 mm and a resolution of 1-15 μm which is sufficient to illustrate structural differences between healthy tissue and brain tumor. Therefore, we investigated gray and white matter of healthy central nervous system and meningioma samples with a Spectral Domain OCT System (Thorlabs Callisto). Additional OCT images were generated after paraffin embedding and after the samples were cut into 10 μm thin slices for histological investigation with a bright field microscope. All samples were stained with Hematoxylin and Eosin. In all cases B-scans and 3D images were made. Furthermore, a camera image of the investigated area was made by the built-in video camera of our OCT system. For orientation, the backsides of all samples were marked with blue ink. The structural differences between healthy tissue and meningioma samples were most pronounced directly after removal. After paraffin embedding these differences diminished. A correlation between OCT en face images and microscopy images can be seen. In order to increase contrast, post processing algorithms were applied. Hence we employed Spectroscopic OCT, pattern recognition algorithms and machine learning algorithms such as k-means Clustering and Principal Component Analysis.

  6. Optimized design and analysis of preclinical intervention studies in vivo

    PubMed Central

    Laajala, Teemu D.; Jumppanen, Mikael; Huhtaniemi, Riikka; Fey, Vidal; Kaur, Amanpreet; Knuuttila, Matias; Aho, Eija; Oksala, Riikka; Westermarck, Jukka; Mäkelä, Sari; Poutanen, Matti; Aittokallio, Tero

    2016-01-01

    Recent reports have called into question the reproducibility, validity and translatability of the preclinical animal studies due to limitations in their experimental design and statistical analysis. To this end, we implemented a matching-based modelling approach for optimal intervention group allocation, randomization and power calculations, which takes full account of the complex animal characteristics at baseline prior to interventions. In prostate cancer xenograft studies, the method effectively normalized the confounding baseline variability, and resulted in animal allocations which were supported by RNA-seq profiling of the individual tumours. The matching information increased the statistical power to detect true treatment effects at smaller sample sizes in two castration-resistant prostate cancer models, thereby leading to saving of both animal lives and research costs. The novel modelling approach and its open-source and web-based software implementations enable the researchers to conduct adequately-powered and fully-blinded preclinical intervention studies, with the aim to accelerate the discovery of new therapeutic interventions. PMID:27480578

  7. In vivo control of soluble guanylate cyclase activation by nitric oxide: a kinetic analysis.

    PubMed Central

    Condorelli, P; George, S C

    2001-01-01

    Free nitric oxide (NO) activates soluble guanylate cyclase (sGC), an enzyme, within both pulmonary and vascular smooth muscle. sGC catalyzes the cyclization of guanosine 5'-triphosphate to guanosine 3',5'-cyclic monophosphate (cGMP). Binding rates of NO to the ferrous heme(s) of sGC have been measured in vitro. However, a missing link in our understanding of the control mechanism of sGC by NO is a comprehensive in vivo kinetic analysis. Available literature data suggests that NO dissociation from the heme center of sGC is accelerated by its interaction with one or more cofactors in vivo. We present a working model for sGC activation and NO consumption in vivo. Our model predicts that NO influences the cGMP formation rate over a concentration range of approximately 5-100 nM (apparent Michaelis constant approximately 23 nM), with Hill coefficients between 1.1 and 1.5. The apparent reaction order for NO consumption by sGC is dependent on NO concentration, and varies between 0 and 1.5. Finally, the activation of sGC (half-life approximately 1-2 s) is much more rapid than deactivation (approximately 50 s). We conclude that control of sGC in vivo is most likely ultra-sensitive, and that activation in vivo occurs at lower NO concentrations than previously reported. PMID:11325714

  8. Detection of low-amplitude in vivo intrinsic signals from an optical imager of retinal function

    NASA Astrophysics Data System (ADS)

    Barriga, Eduardo S.; T'so, Dan; Pattichis, Marios; Kwon, Young; Kardon, Randy; Abramoff, Michael; Soliz, Peter

    2006-02-01

    In the early stages of some retinal diseases, such as glaucoma, loss of retinal activity may be difficult to detect with today's clinical instruments. Many of today's instruments focus on detecting changes in anatomical structures, such as the nerve fiber layer. Our device, which is based on a modified fundus camera, seeks to detect changes in optical signals that reflect functional changes in the retina. The functional imager uses a patterned stimulus at wavelength of 535nm. An intrinsic functional signal is collected at a near infrared wavelength. Measured changes in reflectance in response to the visual stimulus are on the order of 0.1% to 1% of the total reflected intensity level, which makes the functional signal difficult to detect by standard methods because it is masked by other physiological signals and by imaging system noise. In this paper, we analyze the video sequences from a set of 60 experiments with different patterned stimuli from cats. Using a set of statistical techniques known as Independent Component Analysis (ICA), we estimate the signals present in the videos. Through controlled simulation experiments, we quantify the limits of signal strength in order to detect the physiological signal of interest. The results of the analysis show that, in principle, signal levels of 0.1% (-30dB) can be detected. The study found that in 86% of the animal experiments the patterned stimuli effects on the retina can be detected and extracted. The analysis of the different responses extracted from the videos can give an insight of the functional processes present during the stimulation of the retina.

  9. Functional analysis and treatment of diurnal bruxism.

    PubMed

    Lang, Russell; Davenport, Katy; Britt, Courtney; Ninci, Jennifer; Garner, Jennifer; Moore, Melissa

    2013-01-01

    An analogue functional analysis identified attention as a function for a 5-year-old boy's bruxism (teeth grinding). Functional communication training resulted in a reduction of bruxism and an increase in alternative mands for attention. Results were maintained 3 weeks following the intervention.

  10. Functional Analysis and Reduction of Inappropriate Spitting

    ERIC Educational Resources Information Center

    Carter, Stacy L.; Wheeler, John J.

    2007-01-01

    Functional analysis was used to determine the possible function of inappropriate spitting behavior of an adult woman who had been diagnosed with profound mental retardation. Results of an initial descriptive assessment indicated a possible attention function and led to an attention-based intervention, which was deemed ineffective at reducing the…

  11. Functional data analysis in brain imaging studies.

    PubMed

    Tian, Tian Siva

    2010-01-01

    Functional data analysis (FDA) considers the continuity of the curves or functions, and is a topic of increasing interest in the statistics community. FDA is commonly applied to time-series and spatial-series studies. The development of functional brain imaging techniques in recent years made it possible to study the relationship between brain and mind over time. Consequently, an enormous amount of functional data is collected and needs to be analyzed. Functional techniques designed for these data are in strong demand. This paper discusses three statistically challenging problems utilizing FDA techniques in functional brain imaging analysis. These problems are dimension reduction (or feature extraction), spatial classification in functional magnetic resonance imaging studies, and the inverse problem in magneto-encephalography studies. The application of FDA to these issues is relatively new but has been shown to be considerably effective. Future efforts can further explore the potential of FDA in functional brain imaging studies.

  12. An Ex Vivo Model in Human Femoral Heads for Histopathological Study and Resonance Frequency Analysis of Dental Implant Primary Stability

    PubMed Central

    Hernández-Cortés, Pedro; Galindo-Moreno, Pablo; Catena, Andrés; Ortega-Oller, Inmaculada; Salas-Pérez, José; Gómez-Sánchez, Rafael; Aguilar, Mariano; Aguilar, David

    2014-01-01

    Objective. This study was designed to explore relationships of resonance frequency analysis (RFA)—assessed implant stability (ISQ values) with bone morphometric parameters and bone quality in an ex vivo model of dental implants placed in human femoral heads and to evaluate the usefulness of this model for dental implant studies. Material and Methods. This ex vivo study included femoral heads from 17 patients undergoing surgery for femoral neck fracture due to osteoporosis (OP) (n = 7) or for total prosthesis joint replacement due to severe hip osteoarthrosis (OA) (n = 10). Sixty 4.5 × 13 mm Dentsply Astra implants were placed, followed by RFA. CD44 immunohistochemical analysis for osteocytes was also carried out. Results. As expected, the analysis yielded significant effects of femoral head type (OA versus OA) (P < 0.001), but not of the implants (P = 0.455) or of the interaction of the two factors (P = 0.848). Bonferroni post hoc comparisons showed a lower mean ISQ for implants in decalcified (50.33 ± 2.92) heads than in fresh (66.93 ± 1.10) or fixated (70.77 ± 1.32) heads (both P < 0.001). The ISQ score (fresh) was significantly higher for those in OA (73.52 ± 1.92) versus OP (67.13 ± 1.09) heads. However, mixed linear analysis showed no significant association between ISQ scores and morphologic or histomorphometric results (P > 0.5 in all cases), and no significant differences in ISQ values were found as a function of the length or area of the cortical layer (both P > 0.08). Conclusion. Although RFA-determined ISQ values are not correlated with morphometric parameters, they can discriminate bone quality (OP versus OA). This ex vivo model is useful for dental implant studies. PMID:24995307

  13. Functional graphene oxide as a plasmid-based Stat3 siRNA carrier inhibits mouse malignant melanoma growth in vivo

    NASA Astrophysics Data System (ADS)

    Yin, Di; Li, Yang; Lin, Hang; Guo, Baofeng; Du, Yanwei; Li, Xin; Jia, Huijie; Zhao, Xuejian; Tang, Jun; Zhang, Ling

    2013-03-01

    Graphene oxide (GO) has attracted intensive interest in the biomedical field in recent years. We investigate whether the use of functional graphene oxide as an efficient delivery system for delivering specific molecular antitumor therapeutics in vivo could achieve a more excellent antitumor effect. Constitutive activation of signal transducer and activator of transcription 3 (Stat3) promotes survival in a wide spectrum of human cancers. In this paper, we study the in vivo behavior of graphene oxide chemically functionalized with polyethylenimine and polyethylene glycol (GO-PEI-PEG) as a plasmid-based Stat3-specific small interfering RNA (siRNA) carrier in mouse malignant melanoma. The in vivo results indicate significant regression in tumor growth and tumor weight after plasmid-based Stat3 siRNA delivered by GO-PEI-PEG treatment. Moreover, there was no significant side effect from GO-PEI-PEG treatment according to histological examination and blood chemistry analysis in mice. Thus, our work is the first success of using GO-PEI-PEG as a promising carrier for plasmid Stat3 siRNA delivery and down-regulation of Stat3 by a polymer-mediated vehicle and suggests the great promise of graphene in biomedical applications such as cancer treatment.

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

    PubMed

    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.

  15. A Surface Groove Essential for Viral Bcl-2 Function During Chronic Infection In Vivo

    PubMed Central

    Petros, Andrew M; Nettesheim, David; van Dyk, Linda F.; Labrada, Lucia; Speck, Samuel H; Levine, Beth

    2005-01-01

    Antiapoptotic Bcl-2 family proteins inhibit apoptosis in cultured cells by binding BH3 domains of proapoptotic Bcl-2 family members via a hydrophobic BH3 binding groove on the protein surface. We investigated the physiological importance of the BH3 binding groove of an antiapoptotic Bcl-2 protein in mammals in vivo by analyzing a viral Bcl-2 family protein. We show that the γ-herpesvirus 68 (γHV68) Bcl-2 family protein (γHV68 v-Bcl-2), which is known to inhibit apoptosis in cultured cells, inhibits both apoptosis in primary lymphocytes and Bax toxicity in yeast. Nuclear magnetic resonance determination of the γHV68 v-Bcl-2 structure revealed a BH3 binding groove that binds BH3 domain peptides from proapoptotic Bcl-2 family members Bax and Bak via a molecular mechanism shared with host Bcl-2 family proteins, involving a conserved arginine in the BH3 peptide binding groove. Mutations of this conserved arginine and two adjacent amino acids to alanine (SGR to AAA) within the BH3 binding groove resulted in a properly folded protein that lacked the capacity of the wild-type γHV68 v-Bcl-2 to bind Bax BH3 peptide and to block Bax toxicity in yeast. We tested the physiological importance of this v-Bcl-2 domain during viral infection by engineering viral mutants encoding a v-Bcl-2 containing the SGR to AAA mutation. This mutation resulted in a virus defective for both efficient reactivation of γHV68 from latency and efficient persistent γHV68 replication. These studies demonstrate an essential functional role for amino acids in the BH3 peptide binding groove of a viral Bcl-2 family member during chronic infection. PMID:16201011

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

    PubMed Central

    Yin, Li; Chen, Kaining; Guo, Lin; Cheng, Liangjun; Wang, Fuyou; Yang, Liu

    2015-01-01

    Purpose 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. Methods 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. Results 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). Conclusion The CA is closer to the FEA compared with the TEA; it can better serve as an anatomical surrogate for the functional knee axis. PMID:26039711

  17. Sensitivity analysis of near-infrared functional lymphatic imaging

    PubMed Central

    Weiler, Michael; Kassis, Timothy

    2012-01-01

    Abstract. Near-infrared imaging of lymphatic drainage of injected indocyanine green (ICG) has emerged as a new technology for clinical imaging of lymphatic architecture and quantification of vessel function, yet the imaging capabilities of this approach have yet to be quantitatively characterized. We seek to quantify its capabilities as a diagnostic tool for lymphatic disease. Imaging is performed in a tissue phantom for sensitivity analysis and in hairless rats for in vivo testing. To demonstrate the efficacy of this imaging approach to quantifying immediate functional changes in lymphatics, we investigate the effects of a topically applied nitric oxide (NO) donor glyceryl trinitrate ointment. Premixing ICG with albumin induces greater fluorescence intensity, with the ideal concentration being 150  μg/mL ICG and 60  g/L albumin. ICG fluorescence can be detected at a concentration of 150  μg/mL as deep as 6 mm with our system, but spatial resolution deteriorates below 3 mm, skewing measurements of vessel geometry. NO treatment slows lymphatic transport, which is reflected in increased transport time, reduced packet frequency, reduced packet velocity, and reduced effective contraction length. NIR imaging may be an alternative to invasive procedures measuring lymphatic function in vivo in real time. PMID:22734775

  18. Sensitivity analysis of near-infrared functional lymphatic imaging

    NASA Astrophysics Data System (ADS)

    Weiler, Michael; Kassis, Timothy; Dixon, J. Brandon

    2012-06-01

    Near-infrared imaging of lymphatic drainage of injected indocyanine green (ICG) has emerged as a new technology for clinical imaging of lymphatic architecture and quantification of vessel function, yet the imaging capabilities of this approach have yet to be quantitatively characterized. We seek to quantify its capabilities as a diagnostic tool for lymphatic disease. Imaging is performed in a tissue phantom for sensitivity analysis and in hairless rats for in vivo testing. To demonstrate the efficacy of this imaging approach to quantifying immediate functional changes in lymphatics, we investigate the effects of a topically applied nitric oxide (NO) donor glyceryl trinitrate ointment. Premixing ICG with albumin induces greater fluorescence intensity, with the ideal concentration being 150 μg/mL ICG and 60 g/L albumin. ICG fluorescence can be detected at a concentration of 150 μg/mL as deep as 6 mm with our system, but spatial resolution deteriorates below 3 mm, skewing measurements of vessel geometry. NO treatment slows lymphatic transport, which is reflected in increased transport time, reduced packet frequency, reduced packet velocity, and reduced effective contraction length. NIR imaging may be an alternative to invasive procedures measuring lymphatic function in vivo in real time.

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

    NASA Technical Reports Server (NTRS)

    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.

  20. Network Analysis Reveals Similar Transcriptomic Responses to Intrinsic Properties of Carbon Nanomaterials in Vitro and in Vivo.

    PubMed

    Kinaret, Pia; Marwah, Veer; Fortino, Vittorio; Ilves, Marit; Wolff, Henrik; Ruokolainen, Lasse; Auvinen, Petri; Savolainen, Kai; Alenius, Harri; Greco, Dario

    2017-04-11

    Understanding the complex molecular alterations related to engineered nanomaterial (ENM) exposure is essential for carrying out toxicity assessment. Current experimental paradigms rely on both in vitro and in vivo exposure setups that often are difficult to compare, resulting in questioning the real efficacy of cell models to mimic more complex exposure scenarios at the organism level. Here, we have systematically investigated transcriptomic responses of the THP-1 macrophage cell line and lung tissues of mice, after exposure to several carbon nanomaterials (CNMs). Under the assumption that the CNM exposure related molecular alterations are mixtures of signals related to their intrinsic properties, we inferred networks of responding genes, whose expression levels are coordinately altered in response to specific CNM intrinsic properties. We observed only a minute overlap between the sets of intrinsic property-correlated genes at different exposure scenarios, suggesting specific transcriptional programs working in different exposure scenarios. However, when the effects of the CNM were investigated at the level of significantly altered molecular functions, a broader picture of substantial commonality emerged. Our results imply that in vitro exposures can efficiently recapitulate the complex molecular functions altered in vivo. In this study, altered molecular pathways in response to specific CNM intrinsic properties have been systematically characterized from transcriptomic data generated from multiple exposure setups. Our computational approach to the analysis of network response modules further revealed similarities between in vitro and in vivo exposures that could not be detected by traditional analysis of transcriptomics data. Our analytical strategy also opens a possibility to look for pathways of toxicity and understanding the molecular and cellular responses identified across predefined biological themes.

  1. Functional testing of topical skin formulations using an optimised ex vivo skin organ culture model.

    PubMed

    Sidgwick, G P; McGeorge, D; Bayat, A

    2016-07-01

    A number of equivalent-skin models are available for investigation of the ex vivo effect of topical application of drugs and cosmaceuticals onto skin, however many have their drawbacks. With the March 2013 ban on animal models for cosmetic testing of products or ingredients for sale in the EU, their utility for testing toxicity and effect on skin becomes more relevant. The aim of this study was to demonstrate proof of principle that altered expression of key gene and protein markers could be quantified in an optimised whole tissue biopsy culture model. Topical formulations containing green tea catechins (GTC) were investigated in a skin biopsy culture model (n = 11). Punch biopsies were harvested at 3, 7 and 10 days, and analysed using qRT-PCR, histology and HPLC to determine gene and protein expression, and transdermal delivery of compounds of interest. Reduced gene expression of α-SMA, fibronectin, mast cell tryptase, mast cell chymase, TGF-β1, CTGF and PAI-1 was observed after 7 and 10 days compared with treated controls (p < 0.05). Histological analysis indicated a reduction in mast cell tryptase and chymase positive cell numbers in treated biopsies compared with untreated controls at day 7 and day 10 (p < 0.05). Determination of transdermal uptake indicated that GTCs were detected in the biopsies. This model could be adapted to study a range of different topical formulations in both normal and diseased skin, negating the requirement for animal models in this context, prior to study in a clinical trial environment.

  2. Influence of IL-3 functional fragment on cord blood stem cell ex vivo expansion and differentiation

    PubMed Central

    Ren, Zhihua; Zhang, Yu; Zhang, Yanxi; Jiang, Wenhong; Dai, Wei; Ding, Xinxin

    2016-01-01

    Background Recombinant human interleukin-3 (rhIL-3) is a multiple hematopoietic growth factor, which enhances stem cell expansion and hematopoiesis regeneration in vitro and in vivo, when administrated in combination with other cytokines. However, the structure-function study of rhIL-3 remains rarely studied, so far. The purpose of this study was to recognize the short peptide with similar function as rhIL-3, and assess the hematopoietic efficacy in umbilical cord blood (UCB) stem cell culture as well. Methods Two novel monoclonal antibodies (mAb) (C1 and E1) were generated against rhIL-3 using hybridoma technique. Eleven short peptides were depicted and synthesized to overlap covering the full length sequence of rhIL-3. ELISA was employed to distinguish the antibody-binding peptide from the negative peptides. In addition, the multi-potential hematopoiesis capabilities of the positive peptides were evaluated by adding 25 ng/mL of each peptide to the culture medium of hematopoietic stem cells (HSCs) derived from UCB. Total nucleated cell number and the CD34+ cell number from each individual treatment group were calculated on day 7. Correlated antibodies at 0.5 or 2 molar fold to each peptide were also tested in the stem cell expansion experiment, to further confirm the bioactivity of the peptides. Results Two peptides were recognized by the novel generated antibodies, using ELISA. Peptide 3 and 8 exhibited comparable hematopoiesis potentials, with 25.01±0.14 fold, and 19.89±0.12 fold increase of total nucleated cell number on day 7, respectively, compared with the basal medium control (4.93±0.55 fold). These biological effects were neutralized by adding the corresponding mAb at a dose dependent manner. Conclusions Our results identified two specific regions of rhIL-3 responsible for HSC proliferation and differentiation, which were located from 28 to 49 amino acids (P3), and 107 to 127 amino acids (P8), respectively. The short peptide 3 and 8 might act

  3. Functional characterization of the 180-kD ribosome receptor in vivo

    PubMed Central

    1995-01-01

    morphological characterization. Taken together these data provide further evidence that RRp functions as a ribosome receptor in vitro, provide new evidence indicating its functionality in vivo, and in both cases indicate that the NH2-terminal basic domain is essential for ribosome binding. PMID:7790375

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

    PubMed Central

    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. PMID:27725802

  5. Functional data analysis view of functional near infrared spectroscopy data.

    PubMed

    Barati, Zeinab; Zakeri, Issa; Pourrezaei, Kambiz

    2013-11-01

    Functional near infrared spectroscopy (fNIRS) is a powerful tool for the study of oxygenation and hemodynamics of living tissues. Despite the continuous nature of the processes generating the data, analysis of fNIRS data has been limited to discrete-time methods. We propose a technique, namely functional data analysis (fDA), that converts discrete samples to continuous curves. We used fNIRS data collected on forehead during a cold pressor test (CPT) from 20 healthy subjects. Using functional principal component analysis, oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) curves were decomposed into several components based on variability across the subjects. Each component corresponded to an experimental condition and provided qualitative and quantitative information of the shape and weight of that component. Furthermore, we applied functional canonical correlation analysis to investigate the interaction between Hb and HbO2 curves. We showed that the variation of Hb and HbO2 was positively correlated during the CPT, with a "far" channel on right forehead showing a smaller and faster HbO2 variation than Hb. This research suggests the fDA platform for the analysis of fNIRS data, which solves problem of high dimensionality, enables study of response dynamics, enhances characterization of the evoked response, and may improve design of future fNIRS experiments.

  6. Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat.

    PubMed

    Tartaro, Karrie; VanVolkenburg, Maria; Wilkie, Dean; Coskran, Timothy M; Kreeger, John M; Kawabata, Thomas T; Casinghino, Sandra

    2015-01-01

    The mononuclear phagocyte system (MPS) which provides protection against infection is made up of phagocytic cells that engulf and digest bacteria or other foreign substances. Suppression of the MPS may lead to decreased clearance of pathogenic microbes. Drug delivery systems and immunomodulatory therapeutics that target phagocytes have a potential to inhibit MPS function. Available methods to measure inhibition of MPS function use uptake of radioactively-labeled cells or labor-intensive semi-quantitative histologic techniques. The objective of this work was to develop a non-radioactive quantitative method to measure MPS function in vivo by administering heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles(®)). Fluorescence of the Bioparticles(®) is increased at low pH when they are in phagocytic lysosomes. The amount of Bioparticles(®) phagocytosed by MPS organs in rats was determined by measuring fluorescence intensity in livers and spleens ex vivo using an IVIS(®) Spectrum Pre-clinical In Vivo Imaging System. Phagocytosis of the particles by peripheral blood neutrophils was measured by flow cytometry. To assess method sensitivity, compounds likely to suppress the MPS [clodronate-containing liposomes, carboxylate-modified latex particles, maleic vinyl ether (MVE) polymer] were administered to rats prior to injection of the Bioparticles(®). The E. coli particles consistently co-localized with macrophage markers in the liver but not in the spleen. All of the compounds tested decreased phagocytosis in the liver, but had no consistent effects on phagocytic activity in the spleen. In addition, administration of clodronate liposomes and MVE polymer increased the percentage of peripheral blood neutrophils that phagocytosed the Bioparticles(®). In conclusion, an in vivo rat model was developed that measures phagocytosis of E. coli particles in the liver and may be used to assess the impact of test compounds on MPS function. Still, the

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

    SciTech Connect

    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. Here, our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.

  8. Set1 and MLL1/2 target distinct sets of functionally different genomic loci in vivo

    PubMed Central

    Duncan, Elizabeth M.; Chitsazan, Alex D.; Seidel, Chris W.; Alvarado, Alejandro Sánchez

    2015-01-01

    SUMMARY Histone H3 lysine 4 trimethylation (H3K4me3) is known to correlate with both active and poised genomic loci, yet many questions remain regarding its functional roles in vivo. We identify functional genomic targets of two H3K4 methyltransferases, Set1 and MLL1/2, in both the stem cells and differentiated tissue of the planarian flatworm Schmidtea mediterranea. We show that, despite their common substrate, these enzymes target distinct genomic loci in vivo, which are distinguishable by the pattern each enzyme leaves on the chromatin template, i.e., the breadth of the H3K4me3 peak. Whereas Set1 targets are largely associated with the maintenance of the stem cell population, MLL1/2 targets are specifically enriched for genes involved in ciliogenesis. These data not only confirm that chromatin regulation is fundamental to planarian stem cell function, but also provide evidence for post-embryonic functional specificity of H3K4me3 methyltransferases in vivo. PMID:26711341

  9. Lipopolysaccharide (LPS) disrupts particle transport, cilia function and sperm motility in an ex vivo oviduct model

    PubMed Central

    O’Doherty, A. M.; Di Fenza, M.; Kölle, S.

    2016-01-01

    The oviduct functions in the transportation of gametes to the site of fertilization (the ampulla) and is the site of early embryonic development. Alterations of this early developmental environment, such as the presence of sexually transmitted pathogens, may affect oviduct function leading to reduced fertilization rates and contribute to compromised embryonic development. In this study, sperm interactions, particle transport speed (PTS) and cilia beat frequency (CBF) in the ampulla following exposure to lipopolysaccharide (LPS), a constituent of the sexually transmitted pathogens Chlamydia trachomatis and Chlamydia abortus, was investigated. Three complementary experiments were performed to analyse; (1) bound sperm motility and cilia function (2) transport velocity in the oviduct and (3) the expression of genes related to immune function and inflammatory response (CASP3, CD14, MYD88, TLR4 and TRAF6). The motility of bound sperm was significantly lower in ampullae that were exposed to LPS. CBF and PTS significantly increased after treatment with LPS for 2 hours. Finally, gene expression analysis revealed that CASP3 and CD14 were significantly upregulated and TLR4 trended towards increased expression following treatment with LPS. These findings provide an insight on the impact of LPS on the oviduct sperm interaction, and have implications for both male and female fertility. PMID:27079521

  10. In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters.

    PubMed

    Hama, Kotaro; Provost, Elayne; Baranowski, Timothy C; Rubinstein, Amy L; Anderson, Jennifer L; Leach, Steven D; Farber, Steven A

    2009-02-01

    Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae.

  11. Corneal Viscoelastic Properties from Finite-Element Analysis of In Vivo Air-Puff Deformation

    PubMed Central

    Kling, Sabine; Bekesi, Nandor; Dorronsoro, Carlos; Pascual, Daniel; Marcos, Susana

    2014-01-01

    Biomechanical properties are an excellent health marker of biological tissues, however they are challenging to be measured in-vivo. Non-invasive approaches to assess tissue biomechanics have been suggested, but there is a clear need for more accurate techniques for diagnosis, surgical guidance and treatment evaluation. Recently air-puff systems have been developed to study the dynamic tissue response, nevertheless the experimental geometrical observations lack from an analysis that addresses specifically the inherent dynamic properties. In this study a viscoelastic finite element model was built that predicts the experimental corneal deformation response to an air-puff for different conditions. A sensitivity analysis reveals significant contributions to corneal deformation of intraocular pressure and corneal thickness, besides corneal biomechanical properties. The results show the capability of dynamic imaging to reveal inherent biomechanical properties in vivo. Estimates of corneal biomechanical parameters will contribute to the basic understanding of corneal structure, shape and integrity and increase the predictability of corneal surgery. PMID:25121496

  12. How mitochondrial dysfunction affects zebrafish development and cardiovascular function: an in vivo model for testing mitochondria-targeted drugs

    PubMed Central

    Pinho, Brígida R; Santos, Miguel M; Fonseca-Silva, Anabela; Valentão, Patrícia; Andrade, Paula B; Oliveira, Jorge M A

    2013-01-01

    Background and Purpose Mitochondria are a drug target in mitochondrial dysfunction diseases and in antiparasitic chemotherapy. While zebrafish is increasingly used as a biomedical model, its potential for mitochondrial research remains relatively unexplored. Here, we perform the first systematic analysis of how mitochondrial respiratory chain inhibitors affect zebrafish development and cardiovascular function, and assess multiple quinones, including ubiquinone mimetics idebenone and decylubiquinone, and the antimalarial atovaquone. Experimental Approach Zebrafish (Danio rerio) embryos were chronically and acutely exposed to mitochondrial inhibitors and quinone analogues. Concentration-response curves, developmental and cardiovascular phenotyping were performed together with sequence analysis of inhibitor-binding mitochondrial subunits in zebrafish versus mouse, human and parasites. Phenotype rescuing was assessed in co-exposure assays. Key Results Complex I and II inhibitors induced developmental abnormalities, but their submaximal toxicity was not additive, suggesting active alternative pathways for complex III feeding. Complex III inhibitors evoked a direct normal-to-dead transition. ATP synthase inhibition arrested gastrulation. Menadione induced hypochromic anaemia when transiently present following primitive erythropoiesis. Atovaquone was over 1000-fold less lethal in zebrafish than reported for Plasmodium falciparum, and its toxicity partly rescued by the ubiquinone precursor 4-hydroxybenzoate. Idebenone and decylubiquinone delayed rotenone- but not myxothiazol- or antimycin-evoked cardiac dysfunction. Conclusion and Implications This study characterizes pharmacologically induced mitochondrial dysfunction phenotypes in zebrafish, laying the foundation for comparison with future studies addressing mitochondrial dysfunction in this model organism. It has relevant implications for interpreting zebrafish disease models linked to complex I/II inhibition. Further

  13. Hierarchy of neural organization in the embryonic spinal cord: Granger-causality graph analysis of in vivo calcium imaging data.

    PubMed

    Fallani, Fabrizio De Vico; Corazzol, Martina; Sternberg, Jenna R; Wyart, Claire; Chavez, Mario

    2015-05-01

    The recent development of genetically encoded calcium indicators enables monitoring in vivo the activity of neuronal populations. Most analysis of these calcium transients relies on linear regression analysis based on the sensory stimulus applied or the behavior observed. To estimate the basic properties of the functional neural circuitry, we propose a network approach to calcium imaging recorded at single cell resolution. Differently from previous analysis based on cross-correlation, we used Granger-causality estimates to infer information propagation between the activities of different neurons. The resulting functional network was then modeled as a directed graph and characterized in terms of connectivity and node centralities. We applied our approach to calcium transients recorded at low frequency (4 Hz) in ventral neurons of the zebrafish spinal cord at the embryonic stage when spontaneous coiling of the tail occurs. Our analysis on population calcium imaging data revealed a strong ipsilateral connectivity and a characteristic hierarchical organization of the network hubs that supported established propagation of activity from rostral to caudal spinal cord. Our method could be used for detecting functional defects in neuronal circuitry during development and pathological conditions.

  14. Study of Brain Function and Bioenergetics using fMRI and In Vivo MRS at High Fields.

    PubMed

    Chen, Wei

    2005-01-01

    The greatest merit of magnetic resonance (MR) methodology applied to medicine is its capabilities of measuring a variety of physiological parameters in vivo. MR imaging (MRI) with unique imaging contrasts can provide vital information which tightly links to brain functions at both normal and diseased states. In contrast, in vivo MR spectroscopy (MRS) is capable of determining metabolites, bioenergetics and chemical reaction rates in brain noninvasively. These capabilities are further enhanced at high/ultrahigh magnetic fields because of significant gain in MR sensitivity and improvements in the spectral resolution of MRS and imaging contrasts. However, MR research also faces many technical challenges which have attracted many scientists from interdisciplinary research backgrounds to find the optimal solutions. Recent progresses in this research field have showed great promise of MRI/MRS for studying brain function, physiology, and neurochemistry. This talk will discuss the developed MR technologies and their applications in brain study at high fields.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Hyperspectral image classification using functional data analysis.

    PubMed

    Li, Hong; Xiao, Guangrun; Xia, Tian; Tang, Y Y; Li, Luoqing

    2014-09-01

    The large number of spectral bands acquired by hyperspectral imaging sensors allows us to better distinguish many subtle objects and materials. Unlike other classical hyperspectral image classification methods in the multivariate analysis framework, in this paper, a novel method using functional data analysis (FDA) for accurate classification of hyperspectral images has been proposed. The central idea of FDA is to treat multivariate data as continuous functions. From this perspective, the spectral curve of each pixel in the hyperspectral images is naturally viewed as a function. This can be beneficial for making full use of the abundant spectral information. The relevance between adjacent pixel elements in the hyperspectral images can also be utilized reasonably. Functional principal component analysis is applied to solve the classification problem of these functions. Experimental results on three hyperspectral images show that the proposed method can achieve higher classification accuracies in comparison to some state-of-the-art hyperspectral image classification methods.

  17. Computer-aided segmentation and 3D analysis of in vivo MRI examinations of the human vocal tract during phonation

    NASA Astrophysics Data System (ADS)

    Wismüller, Axel; Behrends, Johannes; Hoole, Phil; Leinsinger, Gerda L.; Meyer-Baese, Anke; Reiser, Maximilian F.

    2008-03-01

    We developed, tested, and evaluated a 3D segmentation and analysis system for in vivo MRI examinations of the human vocal tract during phonation. For this purpose, six professionally trained speakers, age 22-34y, were examined using a standardized MRI protocol (1.5 T, T1w FLASH, ST 4mm, 23 slices, acq. time 21s). The volunteers performed a prolonged (>=21s) emission of sounds of the German phonemic inventory. Simultaneous audio tape recording was obtained to control correct utterance. Scans were made in axial, coronal, and sagittal planes each. Computer-aided quantitative 3D evaluation included (i) automated registration of the phoneme-specific data acquired in different slice orientations, (ii) semi-automated segmentation of oropharyngeal structures, (iii) computation of a curvilinear vocal tract midline in 3D by nonlinear PCA, (iv) computation of cross-sectional areas of the vocal tract perpendicular to this midline. For the vowels /a/,/e/,/i/,/o/,/ø/,/u/,/y/, the extracted area functions were used to synthesize phoneme sounds based on an articulatory-acoustic model. For quantitative analysis, recorded and synthesized phonemes were compared, where area functions extracted from 2D midsagittal slices were used as a reference. All vowels could be identified correctly based on the synthesized phoneme sounds. The comparison between synthesized and recorded vowel phonemes revealed that the quality of phoneme sound synthesis was improved for phonemes /a/ and /y/, if 3D instead of 2D data were used, as measured by the average relative frequency shift between recorded and synthesized vowel formants (p<0.05, one-sided Wilcoxon rank sum test). In summary, the combination of fast MRI followed by subsequent 3D segmentation and analysis is a novel approach to examine human phonation in vivo. It unveils functional anatomical findings that may be essential for realistic modelling of the human vocal tract during speech production.

  18. FUNCTIONAL ANALYSIS AND TREATMENT OF COPROPHAGIA

    PubMed Central

    Ing, Anna D; Roane, Henry S; Veenstra, Rebecca A

    2011-01-01

    In the current investigation, functional analysis results suggested that coprophagia, the ingestion of fecal matter, was maintained by automatic reinforcement. Providing noncontingent access to alternative stimuli decreased coprophagia, and the intervention was generalized to two settings. PMID:21541128

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

    PubMed

    Jin, Jun-O; Zhang, Wei; Du, Jiang-Yuan; Wong, Ka-Wing; Oda, Tatsuya; Yu, Qing

    2014-01-01

    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.

  20. A note on population analysis of dissolution-absorption models using the inverse Gaussian function.

    PubMed

    Wang, Jian; Weiss, Michael; D'Argenio, David Z

    2008-06-01

    Because conventional absorption models often fail to describe plasma concentration-time profiles following oral administration, empirical input functions such as the inverse Gaussian function have been successfully used. The purpose of this note is to extend this model by adding a first-order absorption process and to demonstrate the application of population analysis using maximum likelihood estimation via the EM algorithm (implemented in ADAPT 5). In one example, the analysis of bioavailability data of an extended-release formulation, as well as the mean dissolution times estimated in vivo and in vitro with the use of the inverse Gaussian function, is well in accordance, suggesting that the inverse Gaussian function indeed accounts for the in vivo dissolution process. In the other example, the kinetics of trapidil in patients with liver disease, the absorption/dissolution parameters are characterized by a high interindividual variability. Adding a first-order absorption process to the inverse Gaussian function improved the fit in both cases.

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

    PubMed Central

    Lund, Ida K.; Rasch, Morten G.; Ingvarsen, Signe; Pass, Jesper; Madsen, Daniel H.; Engelholm, Lars H.; Behrendt, Niels; Høyer-Hansen, Gunilla

    2012-01-01

    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 protein function(s). This approach has the advantage of being a model for therapeutic targeting. mAbs for use in mouse models can be obtained through immunization of gene-deficient mice with the autologous protein. Such mAbs react with both species-specific epitopes and epitopes conserved between species. mAbs against proteins involved in extracellular proteolysis, including plasminogen activators urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA), their inhibitor PAI-1, the uPA receptor (uPAR), two matrix metalloproteinases (MMP9 and MMP14), as well as the collagen 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 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. PMID:22754528

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

    PubMed

    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.

  3. In Vivo Analysis of Hypertension: Induction of Hypertension, In Vivo Kinase Manipulation, and Assessment of Physiologic Outputs.

    PubMed

    Eguchi, Satoru; Elliott, Katherine

    2017-01-01

    Using an in vivo model system to study signal transduction will include several steps: (1) induce hypertension in the animal, (2) manipulate kinase activation and signal transduction pathways as desired, and (3) observe physiologic outputs. This chapter provides the reader with overviews of the techniques our lab uses to manipulate signal transduction pathways and determine the effects on hypertension.

  4. Functional Analysis and Intervention for Breath Holding.

    ERIC Educational Resources Information Center

    Kern, Lee; And Others

    1995-01-01

    A functional analysis of breath-holding episodes in a 7-year-old girl with severe mental retardation and Cornelia-de-Lange syndrome indicated that breath holding served an operant function, primarily to gain access to attention. Use of extinction, scheduled attention, and a picture card communication system decreased breath holding. (Author/SW)

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

    PubMed

    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.

  6. In vivo analysis of a fluorescent SUMO fusion in transgenic Drosophila

    PubMed Central

    Bocksberger, Marion; Karch, François; Gibert, Jean-Michel

    2014-01-01

    Sumoylation, the covalent attachment of SUMO, a 90 amino acid peptide related to ubiquitin, is a major modulator of protein functions. Fluorescent SUMO protein fusions have been used in cell cultures to visualize SUMO in vivo but not in multicellular organisms. We generated a transgenic line of Drosophila expressing an mCherry-SUMO fusion. We analyzed its pattern in vivo in salivary gland nuclei expressing Venus-HP1 to recognize the different chromatin components (Chromocenter, chromosome IV). We compared it to SUMO immunostaining on squashed polytene chromosomes and observed similar patterns. In addition to the previously reported SUMO localizations (chromosome arms and chromocenter), we identify 2 intense binding sites: the fourth chromosome telomere and the DAPI-bright band in the region 81F. PMID:25483255

  7. A Novel Approach for Studying Histone H1 Function in Vivo

    PubMed Central

    Siriaco, Giorgia; Deuring, Renate; Mawla, Gina D.; Tamkun, John W.

    2015-01-01

    In this report, we investigate the mechanisms that regulate Drosophila histone H1 expression and its association with chromatin in vivo. We show that histone H1 is subject to negative autoregulation and exploit this result to examine the effects of mutations of the main phosphorylation site of histone H1. PMID:25805849

  8. Design and analysis of a novel mechanical loading machine for dynamic in vivo axial loading

    NASA Astrophysics Data System (ADS)

    Macione, James; Nesbitt, Sterling; Pandit, Vaibhav; Kotha, Shiva

    2012-02-01

    This paper describes the construction of a loading machine for performing in vivo, dynamic mechanical loading of the rodent forearm. The loading machine utilizes a unique type of electromagnetic actuator with no mechanically resistive components (servotube), allowing highly accurate loads to be created. A regression analysis of the force created by the actuator with respect to the input voltage demonstrates high linear correlation (R2 = 1). When the linear correlation is used to create dynamic loading waveforms in the frequency (0.5-10 Hz) and load (1-50 N) range used for in vivo loading, less than 1% normalized root mean square error (NRMSE) is computed. Larger NRMSE is found at increased frequencies, with 5%-8% occurring at 40 Hz, and reasons are discussed. Amplifiers (strain gauge, linear voltage displacement transducer (LVDT), and load cell) are constructed, calibrated, and integrated, to allow well-resolved dynamic measurements to be recorded at each program cycle. Each of the amplifiers uses an active filter with cutoff frequency at the maximum in vivo loading frequencies (50 Hz) so that electronic noise generated by the servo drive and actuator are reduced. The LVDT and load cell amplifiers allow evaluation of stress-strain relationships to determine if in vivo bone damage is occurring. The strain gauge amplifier allows dynamic force to strain calibrations to occur for animals of different sex, age, and strain. Unique features are integrated into the loading system, including a weightless mode, which allows the limbs of anesthetized animals to be quickly positioned and removed. Although the device is constructed for in vivo axial bone loading, it can be used within constraints, as a general measurement instrument in a laboratory setting.

  9. Design and analysis of a novel mechanical loading machine for dynamic in vivo axial loading.

    PubMed

    Macione, James; Nesbitt, Sterling; Pandit, Vaibhav; Kotha, Shiva

    2012-02-01

    This paper describes the construction of a loading machine for performing in vivo, dynamic mechanical loading of the rodent forearm. The loading machine utilizes a unique type of electromagnetic actuator with no mechanically resistive components (servotube), allowing highly accurate loads to be created. A regression analysis of the force created by the actuator with respect to the input voltage demonstrates high linear correlation (R(2) = 1). When the linear correlation is used to create dynamic loading waveforms in the frequency (0.5-10 Hz) and load (1-50 N) range used for in vivo loading, less than 1% normalized root mean square error (NRMSE) is computed. Larger NRMSE is found at increased frequencies, with 5%-8% occurring at 40 Hz, and reasons are discussed. Amplifiers (strain gauge, linear voltage displacement transducer (LVDT), and load cell) are constructed, calibrated, and integrated, to allow well-resolved dynamic measurements to be recorded at each program cycle. Each of the amplifiers uses an active filter with cutoff frequency at the maximum in vivo loading frequencies (50 Hz) so that electronic noise generated by the servo drive and actuator are reduced. The LVDT and load cell amplifiers allow evaluation of stress-strain relationships to determine if in vivo bone damage is occurring. The strain gauge amplifier allows dynamic force to strain calibrations to occur for animals of different sex, age, and strain. Unique features are integrated into the loading system, including a weightless mode, which allows the limbs of anesthetized animals to be quickly positioned and removed. Although the device is constructed for in vivo axial bone loading, it can be used within constraints, as a general measurement instrument in a laboratory setting.

  10. Functional Analysis Methodology: Some Closing Comments.

    ERIC Educational Resources Information Center

    Iwata, Brian A.

    1994-01-01

    This commentary summarizes methodological and conceptual issues in functional analysis methodologies and offers some suggestions for their resolution. Considered are increasing complexity, absence of contingencies during assessment, use of subtle manipulations, alternative assessment strategies, analysis of antecedent influences on behavior,…

  11. Functional Analysis and Treatment of Aberrant Behavior.

    ERIC Educational Resources Information Center

    Mace, F. Charles; And Others

    1991-01-01

    This article reviews general classes of variables which help to maintain aberrant behavior including attention seeking, sensory and perceptual consequences, and access to materials or activities. Suggestions for a methodology providing a comprehensive functional analysis are offered which include descriptive analysis, hypothesis forming,…

  12. Diels-Alder functionalized carbon nanotubes for bone tissue engineering: in vitro/in vivo biocompatibility and biodegradability

    NASA Astrophysics Data System (ADS)

    Mata, D.; Amaral, M.; Fernandes, A. J. S.; Colaço, B.; Gama, A.; Paiva, M. C.; Gomes, P. S.; Silva, R. F.; Fernandes, M. H.

    2015-05-01

    The risk-benefit balance for carbon nanotubes (CNTs) dictates their clinical fate. To take a step forward at this crossroad it is compulsory to modulate the CNT in vivo biocompatibility and biodegradability via e.g. chemical functionalization. CNT membranes were functionalised combining a Diels-Alder cycloaddition reaction to generate cyclohexene (-C6H10) followed by a mild oxidisation to yield carboxylic acid groups (-COOH). In vitro proliferation and osteogenic differentiation of human osteoblastic cells were maximized on functionalized CNT membranes (p,f-CNTs). The in vivo subcutaneously implanted materials showed a higher biological reactivity, thus inducing a slighter intense inflammatory response compared to non-functionalized CNT membranes (p-CNTs), but still showing a reduced cytotoxicity profile. Moreover, the in vivo biodegradation of CNTs was superior for p,f-CNT membranes, likely mediated by the oxidation-induced myeloperoxidase (MPO) in neutrophil and macrophage inflammatory milieus. This proves the biodegradability faculty of functionalized CNTs, which potentially avoids long-term tissue accumulation and triggering of acute toxicity. On the whole, the proposed Diels-Alder functionalization accounts for the improved CNT biological response in terms of the biocompatibility and biodegradability profiles. Therefore, CNTs can be considered for use in bone tissue engineering without notable toxicological threats.The risk-benefit balance for carbon nanotubes (CNTs) dictates their clinical fate. To take a step forward at this crossroad it is compulsory to modulate the CNT in vivo biocompatibility and biodegradability via e.g. chemical functionalization. CNT membranes were functionalised combining a Diels-Alder cycloaddition reaction to generate cyclohexene (-C6H10) followed by a mild oxidisation to yield carboxylic acid groups (-COOH). In vitro proliferation and osteogenic differentiation of human osteoblastic cells were maximized on functionalized CNT

  13. In vitro and in vivo analysis of transcription within the replication region of plasmid pIP501.

    PubMed

    Brantl, S; Nuez, B; Behnke, D

    1992-07-01

    Derivatives of the conjugative streptococcal plasmid pIP501 replicate stably in Bacillus subtilis. The region essential for replication of pIP501 has been narrowed down to a 2.2 kb DNA segment, the sequence of which has been determined. This region comprises two genes, copR and repR, proposed to be involved in copy control and replication. By in vitro and in vivo transcriptional analysis we characterized three active promoters, pI, pII and pIII within this region. A putative fourth promoter (pIV) was neither active in vitro nor in vivo. We showed that copR is transcribed from promoter pI while the repR gene is transcribed from promoter pII located just downstream of copR. The pII transcript encompasses a 329 nucleotide (nt) long leader sequence. A counter transcript that was complementary to a major part of this leader was found to originate from a third promoter pIII. The secondary structure of the counter transcript revealed several stem-loop regions. A regulatory function for this antisense RNA in the control of repR expression is proposed. Comparative analysis of the replication regions of pAM beta 1 and pSM19035 suggested a similar organization of transcriptional units, suggesting that an antisense RNA is produced by these plasmids also.

  14. Potent Delivery of Functional Proteins into Mammalian Cells in Vitro and in Vivo Using a Supercharged Protein

    PubMed Central

    2010-01-01

    The inability of proteins to potently penetrate mammalian cells limits their usefulness as tools and therapeutics. When fused to superpositively charged GFP, proteins rapidly (within minutes) entered five different types of mammalian cells with potency up to ∼100-fold greater than that of corresponding fusions with known protein transduction domains (PTDs) including Tat, oligoarginine, and penetratin. Ubiquitin-fused supercharged GFP when incubated with human cells was partially deubiquitinated, suggesting that proteins delivered with supercharged GFP can access the cytosol. Likewise, supercharged GFP delivered functional, nonendosomal recombinase enzyme with greater efficiencies than PTDs in vitro and also delivered functional recombinase enzyme to the retinae of mice when injected in vivo. PMID:20545362

  15. In vivo neutron activation analysis: body composition studies in health and disease

    SciTech Connect

    Ellis, K.J.; Cohn, S.H.

    1984-01-01

    In vivo analysis of body elements by neutron activation is an important tool in medical research. It has provided a direct quantitative measure of body composition of human beings in vivo. Basic physiological differences related to age, sex, race, and body size have been assessed by this noninvasive technique. The diagnosis and management of patients with various metabolic disorders and diseases has also been demonstrated. Two major facilities at Brookhaven are being utilized exclusively for in vivo neutron activation analysis (IVNAA) of calcium, phosphorus, sodium, chlorine, nitrogen, hydrogen, and potassium. These elements serve as the basis for a four compartment model of body composition: protein, water, mineral ash, and fat. Variations in these compartments are demonstrated in clinical research programs investigating obesity, anorexia, cancer, renal failure, osteoporosis, and normal aging. IVNAA continues to provide a unique approach to the evaluation of clinical diagnosis, efficacy of therapeutic regimens, and monitoring of the aging process. Classical balance studies usually require the patient to be admitted to a hospital for extended periods of confinement. IVNAA, however, allows for clinical management of the patient on an out-patient basis, an important aspect for treatment of chronic diseases. 25 references, 3 figures, 5 tables.

  16. A complementation method for functional analysis of mammalian genes

    PubMed Central

    Gonzalez-Santos, Juana Maria; Cao, Huibi; Wang, Anan; Koehler, David R.; Martin, Bernard; Navab, Roya; Hu, Jim

    2005-01-01

    Our progress in understanding mammalian gene function has lagged behind that of gene identification. New methods for mammalian gene functional analysis are needed to accelerate the process. In yeast, the powerful genetic shuffle system allows deletion of any chromosomal gene by homologous recombination and episomal expression of a mutant allele in the same cell. Here, we report a method for mammalian cells, which employs a helper-dependent adenoviral (HD-Ad) vector to synthesize small hairpin (sh) RNAs to knock-down the expression of an endogenous gene by targeting untranslated regions (UTRs). The vector simultaneously expresses an exogenous version of the same gene (wild-type or mutant allele) lacking the UTRs for functional analysis. We demonstrated the utility of the method by using PRPF3, which encodes the human RNA splicing factor Hprp3p. Recently, missense mutations in PRPF3 were found to cause autosomal-dominant Retinitis Pigmentosa, a form of genetic eye diseases affecting the retina. We knocked-down endogenous PRPF3 in multiple cell lines and rescued the phenotype (cell death) with exogenous PRPF3 cDNA, thereby creating a genetic complementation method. Because Ad vectors can efficiently transduce a wide variety of cell types, and many tissues in vivo, this method could have a wide application for gene function studies. PMID:15944448

  17. Relations among Functional Systems in Behavior Analysis

    PubMed Central

    Thompson, Travis

    2007-01-01

    This paper proposes that an organism's integrated repertoire of operant behavior has the status of a biological system, similar to other biological systems, like the nervous, cardiovascular, or immune systems. Evidence from a number of sources indicates that the distinctions between biological and behavioral events is often misleading, engendering counterproductive explanatory controversy. A good deal of what is viewed as biological (often thought to be inaccessible or hypothetical) can become publicly measurable variables using currently available and developing technologies. Moreover, such endogenous variables can serve as establishing operations, discriminative stimuli, conjoint mediating events, and maintaining consequences within a functional analysis of behavior and need not lead to reductionistic explanation. I suggest that explanatory misunderstandings often arise from conflating different levels of analysis and that behavior analysis can extend its reach by identifying variables operating within a functional analysis that also serve functions in other biological systems. PMID:17575907

  18. Coupling of vesicle tethering and Rab binding is required for in vivo functionality of the golgin GMAP-210

    PubMed Central

    Sato, Keisuke; Roboti, Peristera; Mironov, Alexander A.; Lowe, Martin

    2015-01-01

    Golgins are extended coiled-coil proteins believed to participate in membrane-tethering events at the Golgi apparatus. However, the importance of golgin-mediated tethering remains poorly defined, and alternative functions for golgins have been proposed. Moreover, although golgins bind to Rab GTPases, the functional significance of Rab binding has yet to be determined. In this study, we show that depletion of the golgin GMAP-210 causes a loss of Golgi cisternae and accumulation of numerous vesicles. GMAP-210 function in vivo is dependent upon its ability to tether membranes, which is mediated exclusively by the amino-terminal ALPS motif. Binding to Rab2 is also important for GMAP-210 function, although it is dispensable for tethering per se. GMAP-210 length is also functionally important in vivo. Together our results indicate a key role for GMAP-210–mediated membrane tethering in maintaining Golgi structure and support a role for Rab2 binding in linking tethering with downstream docking and fusion events at the Golgi apparatus. PMID:25473115

  19. In vivo skin capacitive imaging analysis by using grey level co-occurrence matrix (GLCM).

    PubMed

    Ou, Xiang; Pan, Wei; Xiao, Perry

    2014-01-02

    We present our latest work on in vivo skin capacitive imaging analysis by using grey level co-occurrence matrix (GLCM). The in vivo skin capacitive images were taken by a capacitance based fingerprint sensor, the skin capacitive images were then analysed by GLCM. Four different GLCM feature vectors, angular second moment (ASM), entropy (ENT), contrast (CON) and correlation (COR), are selected to describe the skin texture. The results show that angular second moment increases as age increases, and entropy decreases as age increases. The results also suggest that the angular second moment values and the entropy values reflect more about the skin texture, whilst the contrast values and the correlation values reflect more about the topically applied solvents. The overall results shows that the GLCM is an effective way to extract and analyse the skin texture information, which can potentially be a valuable reference for evaluating effects of medical and cosmetic treatments.

  20. Hybrid fusions show that inter-monomer electron transfer robustly supports cytochrome bc{sub 1} function in vivo

    SciTech Connect

    Ekiert, Robert; Czapla, Monika; Sarewicz, Marcin; Osyczka, Artur

    2014-08-22

    Highlights: • We used hybrid fusion bc{sub 1} complex to test inter-monomer electron transfer in vivo. • Cross-inactivated complexes were able to sustain photoheterotrophic growth. • Inter-monomer electron transfer supports catalytic cycle in vivo. • bc{sub 1} dimer is functional even when cytochrome b subunits come from different species. - Abstract: Electronic connection between Q{sub o} and Q{sub i} quinone catalytic sites of dimeric cytochrome bc{sub 1} is a central feature of the energy-conserving Q cycle. While both the intra- and inter-monomer electron transfers were shown to connect the sites in the enzyme, mechanistic and physiological significance of the latter remains unclear. Here, using a series of mutated hybrid cytochrome bc{sub 1}-like complexes, we show that inter-monomer electron transfer robustly sustains the function of the enzyme in vivo, even when the two subunits in a dimer come from different species. This indicates that minimal requirement for bioenergetic efficiency is to provide a chain of cofactors for uncompromised electron flux between the catalytic sites, while the details of protein scaffold are secondary.

  1. Novel ex vivo culture method for human monocytes uses shear flow to prevent total loss of transendothelial diapedesis function.

    PubMed

    Tsubota, Yoshiaki; Frey, Jeremy M; Raines, Elaine W

    2014-01-01

    Monocyte recruitment to inflammatory sites and their transendothelial migration into tissues are critical to homeostasis and pathogenesis of chronic inflammatory diseases. However, even short-term suspension culture of primary human monocytes leads to phenotypic changes. In this study, we characterize the functional effects of ex vivo monocyte culture on the steps involved in monocyte transendothelial migration. Our data demonstrate that monocyte diapedesis is impaired by as little as 4 h culture, and the locomotion step is subsequently compromised. After 16 h in culture, monocyte diapedesis is irreversibly reduced by ∼90%. However, maintenance of monocytes under conditions mimicking physiological flow (5-7.5 dyn/cm²) is sufficient to reduce diapedesis impairment significantly. Thus, through the application of shear during ex vivo culture of monocytes, our study establishes a novel protocol, allowing functional analyses of monocytes not currently possible under static culture conditions. These data further suggest that monocyte-based therapeutic applications may be measurably improved by alteration of ex vivo conditions before their use in patients.

  2. Analysis of in vivo mutation data can inform cancer risk assessment.

    PubMed

    Moore, Martha M; Heflich, Robert H; Haber, Lynne T; Allen, Bruce C; Shipp, Annette M; Kodell, Ralph L

    2008-07-01

    Under the new U.S. Environmental Protection Agency (EPA) Cancer Risk Assessment Guidelines [U.S. EPA, 2005. Guidelines for Carcinogen Risk Assessment. EPA/630/P-03/001B, March 2005], the quantitative model chosen for cancer risk assessment is based on the mode-of-action (MOA) of the chemical under consideration. In particular, the risk assessment model depends on whether or not the chemical causes tumors through a direct DNA-reactive mechanism. It is assumed that direct DNA-reactive carcinogens initiate carcinogenesis by inducing mutations and have low-dose linear dose-response curves, whereas carcinogens that operate through a nonmutagenic MOA may have nonlinear dose-responses. We are currently evaluating whether the analysis of in vivo gene mutation data can inform the risk assessment process by better defining the MOA for cancer and thus influencing the choice of the low-dose extrapolation model. This assessment includes both a temporal analysis of mutation induction and a dose-response concordance analysis of mutation with tumor incidence. Our analysis of published data on riddelliine in rats and dichloroacetic acid in mice indicates that our approach has merit. We propose an experimental design and graphical analysis that allow for assessing time-to-mutation and dose-response concordance, thereby optimizing the potential for in vivo mutation data to inform the choice of the quantitative model used in cancer risk assessment.

  3. In vivo elemental analysis by counting neutron-induced gamma rays for medical and biological applications

    NASA Astrophysics Data System (ADS)

    Kehayias, Joseph J.; Ma, Ruimei; Zhuang, Hong; Moore, Robert; Dowling, Lisa

    1995-03-01

    Non-invasive in vivo elemental analysis is a technique used to assess human body composition which is indicative of nutritional status and health condition. The in vivo measurement of the body's major elements is used for a variety of medical studies requiring the determination of the body's compartments (protein, fat, water, bone). Whole body gamma-ray counters, consisting of Nal(Tl) crystal detectors in a shielded room, are used for measuring in vivo the body's Ca, Cl, Na and P by delayed neutron activation analysis. Thermal neutrons from a moderated 238Pu-Be source are used for the measurement of total body nitrogen (and thus protein) and chlorine at low radiation exposure (0.80 mSv). The resulting high energy prompt gamma-rays from nitrogen (10.83 MeV) and chlorine (6.11 MeV) are detected simultaneously with the irradiation. Body fat (the main energy store) and fat distribution (which relates to risk for cardiovascular disease) are measured by detecting C and O in vivo through fast neutron inelastic scattering. A small sealed D-T neutron generator is used for the pulsed (4 - 8 KHz) production of fast neutrons. Carbon and oxygen are detected by counting the 4.44 and 6.13 MeV gamma-rays resulting from the inelastic scattering of the fast neutrons from the 12C and 16O nuclei, respectively. One use of this method is the systematic study of the mechanisms driving the age-associated depletion of the metabolizing, oxygen-consuming cellular compartment of the body. The understanding of this catabolism may suggest ways to maintain lean tissue and thus to preserve quality of life for the very old.

  4. Phenotyping mouse pulmonary function in vivo with the lung diffusing capacity.

    PubMed

    Limjunyawong, Nathachit; Fallica, Jonathan; Ramakrishnan, Amritha; Datta, Kausik; Gabrielson, Matthew; Horton, Maureen; Mitzner, Wayne

    2015-01-06

    The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

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

    PubMed

    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.

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

    PubMed

    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.

  7. High-mobility group box 1 is dispensable for autophagy, mitochondrial quality control, and organ function in vivo.

    PubMed

    Huebener, Peter; Gwak, Geum-Youn; Pradere, Jean-Philippe; Quinzii, Catarina M; Friedman, Richard; Lin, Chyuan-Sheng; Trent, Chad M; Mederacke, Ingmar; Zhao, Enpeng; Dapito, Dianne H; Lin, Yuxi; Goldberg, Ira J; Czaja, Mark J; Schwabe, Robert F

    2014-03-04

    In vitro studies have demonstrated a critical role for high-mobility group box 1 (HMGB1) in autophagy and the autophagic clearance of dysfunctional mitochondria, resulting in severe mitochondrial fragmentation and profound disturbances of mitochondrial respiration in HMGB1-deficient cells. Here, we investigated the effects of HMGB1 deficiency on autophagy and mitochondrial function in vivo, using conditional Hmgb1 ablation in the liver and heart. Unexpectedly, deletion of Hmgb1 in hepatocytes or cardiomyocytes, two cell types with abundant mitochondria, did not alter mitochondrial structure or function, organ function, or long-term survival. Moreover, hepatic autophagy and mitophagy occurred normally in the absence of Hmgb1, and absence of Hmgb1 did not significantly affect baseline and glucocorticoid-induced hepatic gene expression. Collectively, our findings suggest that HMGB1 is dispensable for autophagy, mitochondrial quality control, the regulation of gene expression, and organ function in the adult organism.

  8. In vivo analysis of internal ribosome entry at the Hairless locus by genome engineering in Drosophila.

    PubMed

    Smylla, Thomas K; Preiss, Anette; Maier, Dieter

    2016-10-07

    Cell communication in metazoans requires the highly conserved Notch signaling pathway, which is subjected to strict regulation of both activation and silencing. In Drosophila melanogaster, silencing involves the assembly of a repressor complex by Hairless (H) on Notch target gene promoters. We previously found an in-frame internal ribosome entry site in the full length H transcript resulting in two H protein isoforms (H(p120) and H(p150)). Hence, H may repress Notch signalling activity in situations where cap-dependent translation is inhibited. Here we demonstrate the in vivo importance of both H isoforms for proper fly development. To this end, we replaced the endogenous H locus by constructs specifically affecting translation of either H(p150) or H(p120) isoforms using genome engineering. Our findings indicate the functional relevance of both H proteins. Based on bristle phenotypes, the predominant isoform H(p150) appears to be of particular importance. In contrast, growth regulation and venation of the wing require the concomitant activity of both isoforms. Finally, the IRES dependent production of H(p120) during mitosis was verified in vivo. Together our data confirm IRES mediated translation of H protein in vivo, supporting strict regulation of Notch in different cellular settings.

  9. Noninvasive Strategy Based on Real-Time in Vivo Cataluminescence Monitoring for Clinical Breath Analysis.

    PubMed

    Zhang, Runkun; Huang, Wanting; Li, Gongke; Hu, Yufei

    2017-03-21

    The development of noninvasive methods for real-time in vivo analysis is of great significant, which provides powerful tools for medical research and clinical diagnosis. In the present work, we described a new strategy based on cataluminescence (CTL) for real-time in vivo clinical breath analysis. To illustrate such strategy, a homemade real-time CTL monitoring system characterized by coupling an online sampling device with a CTL sensor for sevoflurane (SVF) was designed, and a real-time in vivo method for the monitoring of SVF in exhaled breath was proposed. The accuracy of the method was evaluated by analyzing the real exhaled breath samples, and the results were compared with those obtained by GC/MS. The measured data obtained by the two methods were in good agreement. Subsequently, the method was applied to real-time monitoring of SVF in exhaled breath from rat models of the control group to investigate elimination pharmacokinetics. In order to further probe the potential of the method for clinical application, the elimination pharmacokinetics of SVF from rat models of control group, liver fibrosis group alcohol liver group, and nonalcoholic fatty liver group were monitored by the method. The raw data of pharmacokinetics of different groups were normalized and subsequently subjected to linear discriminant analysis (LDA). These data were transformed to canonical scores which were visualized as well-clustered with the classification accuracy of 100%, and the overall accuracy of leave-one-out cross-validation procedure is 88%, thereby indicating the utility of the potential of the method for liver disease diagnosis. Our strategy undoubtedly opens up a new door for real-time clinical analysis in a pain-free and noninvasive way and also guides a promising development direction for CTL.

  10. Analysis of the Dynein-Dynactin Interaction In Vitro and In Vivo

    PubMed Central

    King, Stephen J.; Brown, Christa L.; Maier, Kerstin C.; Quintyne, Nicholas J.; Schroer, Trina A.

    2003-01-01

    Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms. PMID:14565986

  11. Inter-laboratory comparison of the in vivo comet assay including three image analysis systems.

    PubMed

    Plappert-Helbig, Ulla; Guérard, Melanie

    2015-12-01

    To compare the extent of potential inter-laboratory variability and the influence of different comet image analysis systems, in vivo comet experiments were conducted using the genotoxicants ethyl methanesulfonate and methyl methanesulfonate. Tissue samples from the same animals were processed and analyzed-including independent slide evaluation by image analysis-in two laboratories with extensive experience in performing the comet assay. The analysis revealed low inter-laboratory experimental variability. Neither the use of different image analysis systems, nor the staining procedure of DNA (propidium iodide vs. SYBR® Gold), considerably impacted the results or sensitivity of the assay. In addition, relatively high stability of the staining intensity of propidium iodide-stained slides was found in slides that were refrigerated for over 3 months. In conclusion, following a thoroughly defined protocol and standardized routine procedures ensures that the comet assay is robust and generates comparable results between different laboratories.

  12. Nano-imaging of the beating mouse heart in vivo: Importance of sarcomere dynamics, as opposed to sarcomere length per se, in the regulation of cardiac function.

    PubMed

    Kobirumaki-Shimozawa, Fuyu; Oyama, Kotaro; Shimozawa, Togo; Mizuno, Akari; Ohki, Takashi; Terui, Takako; Minamisawa, Susumu; Ishiwata, Shin'ichi; Fukuda, Norio

    2016-01-01

    Sarcomeric contraction in cardiomyocytes serves as the basis for the heart's pump functions in mammals. Although it plays a critical role in the circulatory system, myocardial sarcomere length (SL) change has not been directly measured in vivo under physiological conditions because of technical difficulties. In this study, we developed a high speed (100-frames per second), high resolution (20-nm) imaging system for myocardial sarcomeres in living mice. Using this system, we conducted three-dimensional analysis of sarcomere dynamics in left ventricular myocytes during the cardiac cycle, simultaneously with electrocardiogram and left ventricular pressure measurements. We found that (a) the working range of SL was on the shorter end of the resting distribution, and (b) the left ventricular-developed pressure was positively correlated with the SL change between diastole and systole. The present findings provide the first direct evidence for the tight coupling of sarcomere dynamics and ventricular pump functions in the physiology of the heart.

  13. Two functionally distinct domains generated by in vivo cleavage of Nup145p: a novel biogenesis pathway for nucleoporins.

    PubMed Central

    Teixeira, M T; Siniossoglou, S; Podtelejnikov, S; Bénichou, J C; Mann, M; Dujon, B; Hurt, E; Fabre, E

    1997-01-01

    Nup145p is an essential yeast nucleoporin involved in nuclear export of polyadenylated RNAs. We demonstrate here that Nup145p is cleaved in vivo to yield two functionally distinct domains: a carboxy-terminal domain (C-Nup145p) which is located at the nuclear pore complex (NPC) and assembles into the Nup84p complex, and a GLFG-containing amino-terminal domain (N-Nup145p) which is not part of this complex. Whereas the essential C-Nup145p accomplishes the functions required for efficient mRNA export and normal NPC distribution, N-Nup145p, which is homologous to the GLFG-containing nucleoporins Nup100p and Nup116p, is not necessary for cell growth. However, the N-Nup145p becomes essential in a nup188 mutant background. Strikingly, generation of a free N-domain is a prerequisite for complementation of this peculiar synthetic lethal mutant. These data suggest that N- and C-domains of Nup145p perform independent functions, and that the in vivo cleavage observed is of functional importance. PMID:9305650

  14. TRAF binding is required for a distinct subset of in vivo B cell functions of the oncoprotein LMP1.

    PubMed

    Arcipowski, Kelly M; Bishop, Gail A

    2012-12-01

    EBV-encoded latent membrane protein 1 (LMP1) is important for EBV contributions to B cell transformation and many EBV-associated malignancies, as well as EBV-mediated exacerbation of autoimmunity. LMP1 functionally mimics TNF receptor (TNFR) superfamily member CD40, but LMP1 signals and downstream effects are amplified and sustained compared with CD40. CD40 and LMP1 both use TNFR-associated factor (TRAF) adaptor proteins, but in distinct ways. LMP1 functions require TRAFs 3, 5, and 6, which interact with LMP1. However, TRAFs can also contribute to signaling in the absence of direct interactions with cell surface receptors, so we investigated whether their roles in LMP1 in vivo functions require direct association. We show in this study that the LMP1 TRAF binding site was required for LMP1-mediated autoantibody production, the germinal center response to immunization, and optimal production of several isotypes of Ig, but not LMP1-dependent enlargement of secondary lymphoid organs in transgenic mice. Thus, LMP1 in vivo effects can be mediated via both TRAF binding-dependent and -independent pathways. Together with our previous findings, these results indicate that TRAF-dependent receptor functions may not always require TRAF-receptor binding. These data suggest that TRAF-mediated signaling pathways, such as those of LMP1, may be more diverse than previously appreciated. This finding has significant implications for receptor and TRAF-targeted therapies.

  15. Hyperspectral wide gap second derivative analysis for in vivo detection of cervical intraepithelial neoplasia

    NASA Astrophysics Data System (ADS)

    Zheng, Wenli; Wang, Chaojian; Chang, Shufang; Zhang, Shiwu; Xu, Ronald X.

    2015-12-01

    Hyperspectral reflectance imaging technique has been used for in vivo detection of cervical intraepithelial neoplasia. However, the clinical outcome of this technique is suboptimal owing to multiple limitations such as nonuniform illumination, high-cost and bulky setup, and time-consuming data acquisition and processing. To overcome these limitations, we acquired the hyperspectral data cube in a wavelength ranging from 600 to 800 nm and processed it by a wide gap second derivative analysis method. This method effectively reduced the image artifacts caused by nonuniform illumination and background absorption. Furthermore, with second derivative analysis, only three specific wavelengths (620, 696, and 772 nm) are needed for tissue classification with optimal separability. Clinical feasibility of the proposed image analysis and classification method was tested in a clinical trial where cervical hyperspectral images from three patients were used for classification analysis. Our proposed method successfully classified the cervix tissue into three categories of normal, inflammation and high-grade lesion. These classification results were coincident with those by an experienced gynecology oncologist after applying acetic acid. Our preliminary clinical study has demonstrated the technical feasibility for in vivo and noninvasive detection of cervical neoplasia without acetic acid. Further clinical research is needed in order to establish a large-scale diagnostic database and optimize the tissue classification technique.

  16. Multilevel sparse functional principal component analysis.

    PubMed

    Di, Chongzhi; Crainiceanu, Ciprian M; Jank, Wolfgang S

    2014-01-29

    We consider analysis of sparsely sampled multilevel functional data, where the basic observational unit is a function and data have a natural hierarchy of basic units. An example is when functions are recorded at multiple visits for each subject. Multilevel functional principal component analysis (MFPCA; Di et al. 2009) was proposed for such data when functions are densely recorded. Here we consider the case when functions are sparsely sampled and may contain only a few observations per function. We exploit the multilevel structure of covariance operators and achieve data reduction by principal component decompositions at both between and within subject levels. We address inherent methodological differences in the sparse sampling context to: 1) estimate the covariance operators; 2) estimate the functional principal component scores; 3) predict the underlying curves. Through simulations the proposed method is able to discover dominating modes of variations and reconstruct underlying curves well even in sparse settings. Our approach is illustrated by two applications, the Sleep Heart Health Study and eBay auctions.

  17. Analysis of Ventricular Function by Computed Tomography

    PubMed Central

    Rizvi, Asim; Deaño, Roderick C.; Bachman, Daniel P.; Xiong, Guanglei; Min, James K.; Truong, Quynh A.

    2014-01-01

    The assessment of ventricular function, cardiac chamber dimensions and ventricular mass is fundamental for clinical diagnosis, risk assessment, therapeutic decisions, and prognosis in patients with cardiac disease. Although cardiac computed tomography (CT) is a noninvasive imaging technique often used for the assessment of coronary artery disease, it can also be utilized to obtain important data about left and right ventricular function and morphology. In this review, we will discuss the clinical indications for the use of cardiac CT for ventricular analysis, review the evidence on the assessment of ventricular function compared to existing imaging modalities such cardiac MRI and echocardiography, provide a typical cardiac CT protocol for image acquisition and post-processing for ventricular analysis, and provide step-by-step instructions to acquire multiplanar cardiac views for ventricular assessment from the standard axial, coronal, and sagittal planes. Furthermore, both qualitative and quantitative assessments of ventricular function as well as sample reporting are detailed. PMID:25576407

  18. Quantitative analysis of intrinsic skin aging in dermal papillae by in vivo harmonic generation microscopy

    PubMed Central

    Liao, Yi-Hua; Kuo, Wei-Cheng; Chou, Sin-Yo; Tsai, Cheng-Shiun; Lin, Guan-Liang; Tsai, Ming-Rung; Shih, Yuan-Ta; Lee, Gwo-Giun; Sun, Chi-Kuang

    2014-01-01

    Chronological skin aging is associated with flattening of the dermal-epidermal junction (DEJ), but to date no quantitative analysis focusing on the aging changes in the dermal papillae (DP) has been performed. The aim of the study is to determine the architectural changes and the collagen density related to chronological aging in the dermal papilla zone (DPZ) by in vivo harmonic generation microscopy (HGM) with a sub-femtoliter spatial resolution. We recruited 48 Asian subjects and obtained in vivo images on the sun-protected volar forearm. Six parameters were defined to quantify 3D morphological changes of the DPZ, which we analyzed both manually and computationally to study their correlation with age. The depth of DPZ, the average height of isolated DP, and the 3D interdigitation index decreased with age, while DP number density, DP volume, and the collagen density in DP remained constant over time. In vivo high-resolution HGM technology has uncovered chronological aging-related variations in DP, and sheds light on real-time quantitative skin fragility assessment and disease diagnostics based on collagen density and morphology. PMID:25401037

  19. Towards Therapeutic Delivery of Extracellular Vesicles: Strategies for In Vivo Tracking and Biodistribution Analysis

    PubMed Central

    Di Rocco, Giuliana; Baldari, Silvia

    2016-01-01

    Extracellular vesicles (EVs), such as microvesicles and exosomes, are membranous structures containing bioactive material released by several cells types, including mesenchymal stem/stromal cells (MSCs). Increasing lines of evidences point to EVs as paracrine mediators of the beneficial effects on tissue remodeling associated with cell therapy. Administration of MSCs-derived EVs has therefore the potential to open new and safer therapeutic avenues, alternative to cell-based approaches, for degenerative diseases. However, an enhanced knowledge about in vivo EVs trafficking upon delivery is required before effective clinical translation. Only a few studies have focused on the biodistribution analysis of exogenously administered MSCs-derived EVs. Nevertheless, current strategies for in vivo tracking in animal models have provided valuable insights on the biodistribution upon systemic delivery of EVs isolated from several cellular sources, indicating in liver, spleen, and lungs the preferential target organs. Different strategies for targeting EVs to specific tissues to enhance their therapeutic efficacy and reduce possible off-target effects have been investigated. Here, in the context of a possible clinical application of MSC-derived EVs for tissue regeneration, we review the existing strategies for in vivo tracking and targeting of EVs isolated from different cellular sources and the studies elucidating the biodistribution of exogenously administered EVs. PMID:27994623

  20. Functional analysis and intervention for chronic rumination.

    PubMed

    Woods, Kathryn E; Luiselli, James K; Tomassone, Shanon

    2013-01-01

    We conducted a functional analysis and treatment evaluation of chronic rumination in a 19-year-old man with intellectual disabilities. Outcomes of the functional analysis suggested that rumination was maintained by automatic reinforcement. Results of the intervention evaluation suggested that (a) noncontingent access to food after meals reduced rumination more effectively than did noncontingent access to inedible stimuli, (b) a particular type of food was associated with lower levels of rumination than other types of food, and (c) both presession and continuous access to food reduced levels of rumination more effectively than did fixed-time access to food.

  1. Functional analysis of colonic bacterial metabolism: relevant to health?

    PubMed

    Hamer, Henrike M; De Preter, Vicky; Windey, Karen; Verbeke, Kristin

    2012-01-01

    With the use of molecular techniques, numerous studies have evaluated the composition of the intestinal microbiota in health and disease. However, it is of major interest to supplement this with a functional analysis of the microbiota. In this review, the different approaches that have been used to characterize microbial metabolites, yielding information on the functional end products of microbial metabolism, have been summarized. To analyze colonic microbial metabolites, the most conventional way is by application of a hypothesis-driven targeted approach, through quantification of selected metabolites from carbohydrate (e.g., short-chain fatty acids) and protein fermentation (e.g., p-cresol, phenol, ammonia, or H(2)S), secondary bile acids, or colonic enzymes. The application of stable isotope-labeled substrates can provide an elegant solution to study these metabolic pathways in vivo. On the other hand, a top-down approach can be followed by applying metabolite fingerprinting techniques based on (1)H-NMR or mass spectrometric analysis. Quantification of known metabolites and characterization of metabolite patterns in urine, breath, plasma, and fecal samples can reveal new pathways and give insight into physiological regulatory processes of the colonic microbiota. In addition, specific metabolic profiles can function as a diagnostic tool for the identification of several gastrointestinal diseases, such as ulcerative colitis and Crohn's disease. Nevertheless, future research will have to evaluate the relevance of associations between metabolites and different disease states.

  2. Functional analysis of an olfactory receptor in Drosophila melanogaster

    PubMed Central

    Störtkuhl, Klemens F.; Kettler, Raffael

    2001-01-01

    Fifty nine candidate olfactory receptor (Or) genes have recently been identified in Drosophila melanogaster, one of which is Or43a. In wild-type flies, Or43a is expressed at the distal edge of the third antennal segment in about 15 Or neurons. To identify ligands for the receptor we used the Gal4/UAS system to misexpress Or43a in the third antennal segment. Or43a mRNA expression in the antenna of transformed and wild-type flies was visualized by in situ hybridization with a digoxigenin-labeled probe. Electroantennogram recordings from transformed and wild-type flies were used to identify cyclohexanol, cyclohexanone, benzaldehyde, and benzyl alcohol as ligands for the Or43a. This in vivo analysis reveals functional properties of one member of the recently isolated Or family in Drosophila and will provide further insight into our understanding of olfactory coding. PMID:11481495

  3. RNA Enrichment Method for Quantitative Transcriptional Analysis of Pathogens In Vivo Applied to the Fungus Candida albicans

    PubMed Central

    Amorim-Vaz, Sara; Tran, Van Du T.; Pradervand, Sylvain; Pagni, Marco; Coste, Alix T.

    2015-01-01

    ABSTRACT In vivo transcriptional analyses of microbial pathogens are often hampered by low proportions of pathogen biomass in host organs, hindering the coverage of full pathogen transcriptome. We aimed to address the transcriptome profiles of Candida albicans, the most prevalent fungal pathogen in systemically infected immunocompromised patients, during systemic infection in different hosts. We developed a strategy for high-resolution quantitative analysis of the C. albicans transcriptome directly from early and late stages of systemic infection in two different host models, mouse and the insect Galleria mellonella. Our results show that transcriptome sequencing (RNA-seq) libraries were enriched for fungal transcripts up to 1,600-fold using biotinylated bait probes to capture C. albicans sequences. This enrichment biased the read counts of only ~3% of the genes, which can be identified and removed based on a priori criteria. This allowed an unprecedented resolution of C. albicans transcriptome in vivo, with detection of over 86% of its genes. The transcriptional response of the fungus was surprisingly similar during infection of the two hosts and at the two time points, although some host- and time point-specific genes could be identified. Genes that were highly induced during infection were involved, for instance, in stress response, adhesion, iron acquisition, and biofilm formation. Of the in vivo-regulated genes, 10% are still of unknown function, and their future study will be of great interest. The fungal RNA enrichment procedure used here will help a better characterization of the C. albicans response in infected hosts and may be applied to other microbial pathogens. PMID:26396240

  4. Dose-response analysis of heavy metal toxicants in man. Direct in vivo assessment of body burden

    SciTech Connect

    Ellis, K.J.

    1985-06-01

    Differences in uptake, metabolism, and excretion of heavy metals makes selection of a suitable biological media as a monitor of body burden very difficult. Exposure assessments based on body fluid levels can provide, at best, only general population estimates. The most frequently monitored media are blood, urine, nail or hair clippings, sweat, and saliva. Unfortunately each of these tissues can be influenced by recent exposure conditions and are not accurate indices of the total dose or body burden. However, direct in vivo measurements of body burden in humans, have recently been performed. This nuclear technique has focused on the measurements of kidney and liver cadmium (Cd) by neutron activation analysis and bone lead (Pb) determinations using x-ray fluorescence. The dose-response relationship for renal dysfunction based on the direct in vivo body burden for Cd is presented. The most probable Cd value for the kidney associated with renal impairment is approximately 35 mg. Approximately 10% of the subjects with 20 mg Cd in the kidney will have moderately elevated ..beta../sub 2/-microglobulin, an early indicator of potential renal functional changes. 11 refs., 5 figs., 2 tabs.

  5. Medical applications of in vivo neutron inelastic scattering and neutron activation analysis: Technical similarities to detection of explosives and contraband

    NASA Astrophysics Data System (ADS)

    Kehayias, J. J.

    2001-07-01

    Nutritional status of patients can be evaluated by monitoring changes in elemental body composition. Fast neutron activation (for N and P) and neutron inelastic scattering (for C and O) are used in vivo to assess elements characteristic of specific body compartments. There are similarities between the body composition techniques and the detection of hidden explosives and narcotics. All samples have to be examined in depth and the ratio of elements provides a "signature" of the chemical of interest. The N/H and C/O ratios measure protein and fat content in the body. Similarly, a high C/O ratio is characteristic of narcotics and a low C/O together with a strong presence of N is a signature of some explosives. The available time for medical applications is about 20 min—compared to a few seconds for the detection of explosives—but the permitted radiation exposure is limited. In vivo neutron analysis is used to measure H, O, C, N, P, Na, Cl, and Ca for the study of the mechanisms of lean tissue depletion with aging and wasting diseases, and to investigate methods of preserving function and quality of life in the elderly.

  6. A factor analysis model for functional genomics

    PubMed Central

    Kustra, Rafal; Shioda, Romy; Zhu, Mu

    2006-01-01

    Background Expression array data are used to predict biological functions of uncharacterized genes by comparing their expression profiles to those of characterized genes. While biologically plausible, this is both statistically and computationally challenging. Typical approaches are computationally expensive and ignore correlations among expression profiles and functional categories. Results We propose a factor analysis model (FAM) for functional genomics and give a two-step algorithm, using genome-wide expression data for yeast and a subset of Gene-Ontology Biological Process functional annotations. We show that the predictive performance of our method is comparable to the current best approach while our total computation time was faster by a factor of 4000. We discuss the unique challenges in performance evaluation of algorithms used for genome-wide functions genomics. Finally, we discuss extensions to our method that can incorporate the inherent correlation structure of the functional categories to further improve predictive performance. Conclusion Our factor analysis model is a computationally efficient technique for functional genomics and provides a clear and unified statistical framework with potential for incorporating important gene ontology information to improve predictions. PMID:16630343

  7. In vivo Prompt Gamma Neutron Activation Analysis Facility for Total Body Nitrogen and Cd

    SciTech Connect

    Munive, Marco; Revilla, Angel; Solis, Jose L.

    2007-10-26

    A Prompt Gamma Neutron Activation Analysis (PGNAA) system has been designed and constructed to measure the total body nitrogen and Cd for in vivo studies. An aqueous solution of KNO{sub 3} was used as phantom for system calibration. The facility has been used to monitor total body nitrogen (TBN) of mice and found that is related to their diet. Some mice swallowed diluted water with Cl{sub 2}Cd, and the presence of Cd was detected in the animals. The minimum Cd concentration that the system can detect was 20 ppm.

  8. Surface loop resonator design for in vivo EPR tooth dosimetry using finite element analysis.

    PubMed

    Pollock, Jennifer D; Williams, Benjamin B; Sidabras, Jason W; Grinberg, Oleg; Salikhov, Ildar; Lesniewski, Piotr; Kmiec, Maciej; Swartz, Harold M

    2010-02-01

    Finite element analysis is used to evaluate and design L-band surface loop resonators for in vivo electron paramagnetic resonance (EPR) tooth dosimetry. This approach appears to be practical and useful for the systematic examination and evaluation of resonator configurations to enhance the precision of dose estimates. The effects of loop positioning in the mouth are examined, and it is shown that the sensitivity to loop position along a row of molars is decreased as the loop is moved away from the teeth.

  9. Using Bayesian analysis in repeated preclinical in vivo studies for a more effective use of animals.

    PubMed

    Walley, Rosalind; Sherington, John; Rastrick, Joe; Detrait, Eric; Hanon, Etienne; Watt, Gillian

    2016-05-01

    Whilst innovative Bayesian approaches are increasingly used in clinical studies, in the preclinical area Bayesian methods appear to be rarely used in the reporting of pharmacology data. This is particularly surprising in the context of regularly repeated in vivo studies where there is a considerable amount of data from historical control groups, which has potential value. This paper describes our experience with introducing Bayesian analysis for such studies using a Bayesian meta-analytic predictive approach. This leads naturally either to an informative prior for a control group as part of a full Bayesian analysis of the next study or using a predictive distribution to replace a control group entirely. We use quality control charts to illustrate study-to-study variation to the scientists and describe informative priors in terms of their approximate effective numbers of animals. We describe two case studies of animal models: the lipopolysaccharide-induced cytokine release model used in inflammation and the novel object recognition model used to screen cognitive enhancers, both of which show the advantage of a Bayesian approach over the standard frequentist analysis. We conclude that using Bayesian methods in stable repeated in vivo studies can result in a more effective use of animals, either by reducing the total number of animals used or by increasing the precision of key treatment differences. This will lead to clearer results and supports the "3Rs initiative" to Refine, Reduce and Replace animals in research. Copyright © 2016 John Wiley & Sons, Ltd.

  10. Amnionless function is required for cubilin brush-border expression and intrinsic factor-cobalamin (vitamin B12) absorption in vivo.

    PubMed

    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.

  11. Beginner's luck--the first in vivo demonstration of functioning platelets; William Duke, 1910.

    PubMed

    Boulton, F

    2012-04-01

    Blood platelets remained obscure until the early 20th century although from the 1880 s claims that low numbers were associated with certain types of 'purpura' began to gain favour. This article re-appraises critically, but with due consideration to the limited technology of the times, the first remarkable in vivo demonstration of the effects of platelets demonstrated by the serial 'Bleeding Times' reported by William Duke in 1910, when fresh blood was transfused to two thrombocytopenic people. It also speculates on the possible causes of the thrombocytopenia with which Duke's main patient presented.

  12. Simple and effective exercise design for assessing in vivo mitochondrial function in clinical applications using 31P magnetic resonance spectroscopy

    PubMed Central

    Sleigh, Alison; Lupson, Victoria; Thankamony, Ajay; Dunger, David B.; Savage, David B.; Carpenter, T. Adrian; Kemp, Graham J.

    2016-01-01

    The growing recognition of diseases associated with dysfunction of mitochondria poses an urgent need for simple measures of mitochondrial function. Assessment of the kinetics of replenishment of the phosphocreatine pool after exercise using 31P magnetic resonance spectroscopy can provide an in vivo measure of mitochondrial function; however, the wider application of this technique appears limited by complex or expensive MR-compatible exercise equipment and protocols not easily tolerated by frail participants or those with reduced mental capacity. Here we describe a novel in-scanner exercise method which is patient-focused, inexpensive, remarkably simple and highly portable. The device exploits an MR-compatible high-density material (BaSO4) to form a weight which is attached directly to the ankle, and a one-minute dynamic knee extension protocol produced highly reproducible measurements of post-exercise PCr recovery kinetics in both healthy subjects and patients. As sophisticated exercise equipment is unnecessary for this measurement, our extremely simple design provides an effective and easy-to-implement apparatus that is readily translatable across sites. Its design, being tailored to the needs of the patient, makes it particularly well suited to clinical applications, and we argue the potential of this method for investigating in vivo mitochondrial function in new cohorts of growing clinical interest. PMID:26751849

  13. In vivo assessment of cardiac metabolism and function in the abdominal aortic banding model of compensated cardiac hypertrophy

    PubMed Central

    Seymour, Anne-Marie L.; Giles, Lucia; Ball, Vicky; Miller, Jack J.; Clarke, Kieran; Carr, Carolyn A.; Tyler, Damian J.

    2015-01-01

    Aims Left ventricular hypertrophy is an adaptive response of the heart to chronic mechanical overload and can lead to functional deterioration and heart failure. Changes in cardiac energy metabolism are considered as key to the hypertrophic remodelling process. The concurrence of obesity and hypertrophy has been associated with contractile dysfunction, and this work therefore aimed to investigate the in vivo structural, functional, and metabolic remodelling that occurs in the hypertrophied heart in the setting of a high-fat, high-sucrose, Western diet (WD). Methods and results Following induction of cardiac hypertrophy through abdominal aortic banding, male Sprague Dawley rats were exposed to either a standard diet or a WD (containing 45% fat and 16% sucrose) for up to 14 weeks. Cardiac structural and functional characteristics were determined by CINE MRI, and in vivo metabolism was investigated using hyperpolarized 13C-labelled pyruvate. Cardiac hypertrophy was observed at all time points, irrespective of dietary manipulation, with no evidence of cardiac dysfunction. Pyruvate dehydrogenase flux was unchanged in the hypertrophied animals at any time point, but increased incorporation of the 13C label into lactate was observed by 9 weeks and maintained at 14 weeks, indicative of enhanced glycolysis. Conclusion Hypertrophied hearts revealed little evidence of a switch towards increased glucose oxidation but rather an uncoupling of glycolytic metabolism from glucose oxidation. This was maintained under conditions of dietary stress provided by a WD but, at this compensated phase of hypertrophy, did not result in any contractile dysfunction. PMID:25750189

  14. Methods: implementation of in vitro and ex vivo phagocytosis and respiratory burst function assessments in safety testing.

    PubMed

    Freebern, Wendy J; Bigwarfe, Tammy J; Price, Karen D; Haggerty, Helen G

    2013-01-01

    Functional innate immune assessments, including phagocytosis and respiratory burst, are at the forefront of immunotoxicology evaluation in pre-clinical animal species. Although in the clinic and in academic science, phagocytosis, and respiratory burst assessments have been reported for over two decades, the implementation of phagocytosis and respiratory burst analyses in toxicology safety programs is just recently gaining publicity. Discussed herein are general methods, both microtiter plate-based and flow cytometric-based, for assessing phagocytosis and respiratory burst in pre-clinical species including mouse, rat, dog, and monkey. This methods-centric discussion includes a review of technologies and descriptions of method applications, with examples of results from analyses testing reported inhibitors (rottlerin, wortmannin, and SB203580) of phagocytosis and respiratory burst. Justification of implementation, strategic experimental design planning, and feasibility aspects of evaluating test article effects on phagocytosis and respiratory burst function are described within the context of a case study. The case study involves investigation of the effects of a small molecule p38 kinase inhibitor, BMS-582949, on phagocytosis and respiratory burst functions in rat and monkey neutrophils and monocytes in vitro, as well as ex vivo in these innate immune cells from monkeys administered BMS-582949 during a 1-week repeat dose investigative study. The results of the in vitro and ex vivo assessments demonstrated that BMS-582949 inhibited phagocytosis and respiratory burst. These findings correlated with incidences of opportunistic infections observed in rat and monkey toxicity studies.

  15. Solid-phase microextraction technology for in vitro and in vivo metabolite analysis

    PubMed Central

    Zhang, Qihui; Zhou, Liandi; Chen, Hua; Wang, Chong-Zhi; Xia, Zhining; Yuan, Chun-Su

    2016-01-01

    Analysis of endogenous metabolites in biological samples may lead to the identification of biomarkers in metabolomics studies. To achieve accurate sample analysis, a combined method of continuous quick sampling and extraction is required for online compound detection. Solid-phase microextraction (SPME) integrates sampling, extraction and concentration into a single solvent-free step for chemical analysis. SPME has a number of advantages, including simplicity, high sensitivity and a relatively non-invasive nature. In this article, we reviewed SPME technology in in vitro and in vivo analyses of metabolites after the ingestion of herbal medicines, foods and pharmaceutical agents. The metabolites of microorganisms in dietary supplements and in the gastrointestinal tract will also be examined. As a promising technology in biomedical and pharmaceutical research, SPME and its future applications will depend on advances in analytical technologies and material science. PMID:27695152

  16. GPU accelerated dynamic functional connectivity analysis for functional MRI data.

    PubMed

    Akgün, Devrim; Sakoğlu, Ünal; Esquivel, Johnny; Adinoff, Bryon; Mete, Mutlu

    2015-07-01

    Recent advances in multi-core processors and graphics card based computational technologies have paved the way for an improved and dynamic utilization of parallel computing techniques. Numerous applications have been implemented for the acceleration of computationally-intensive problems in various computational science fields including bioinformatics, in which big data problems are prevalent. In neuroimaging, dynamic functional connectivity (DFC) analysis is a computationally demanding method used to investigate dynamic functional interactions among different brain regions or networks identified with functional magnetic resonance imaging (fMRI) data. In this study, we implemented and analyzed a parallel DFC algorithm based on thread-based and block-based approaches. The thread-based approach was designed to parallelize DFC computations and was implemented in both Open Multi-Processing (OpenMP) and Compute Unified Device Architecture (CUDA) programming platforms. Another approach developed in this study to better utilize CUDA architecture is the block-based approach, where parallelization involves smaller parts of fMRI time-courses obtained by sliding-windows. Experimental results showed that the proposed parallel design solutions enabled by the GPUs significantly reduce the computation time for DFC analysis. Multicore implementation using OpenMP on 8-core processor provides up to 7.7× speed-up. GPU implementation using CUDA yielded substantial accelerations ranging from 18.5× to 157× speed-up once thread-based and block-based approaches were combined in the analysis. Proposed parallel programming solutions showed that multi-core processor and CUDA-supported GPU implementations accelerated the DFC analyses significantly. Developed algorithms make the DFC analyses more practical for multi-subject studies with more dynamic analyses.

  17. 4PD Functionalized Dendrimers: A Flexible Tool for In Vivo Gene Silencing of Tumor-Educated Myeloid Cells.

    PubMed

    Zilio, Serena; Vella, Jennifer L; De la Fuente, Adriana C; Daftarian, Pirouz M; Weed, Donald T; Kaifer, Angel; Marigo, Ilaria; Leone, Kevin; Bronte, Vincenzo; Serafini, Paolo

    2017-04-10

    Myeloid cells play a key role in tumor progression and metastasis by providing nourishment and immune protection, as well as facilitating cancer invasion and seeding to distal sites. Although advances have been made in understanding the biology of these tumor-educated myeloid cells (TEMCs), their intrinsic plasticity challenges our further understanding of their biology. Indeed, in vitro experiments only mimic the in vivo setting, and current gene-knockout technologies do not allow the simultaneous, temporally controlled, and cell-specific silencing of multiple genes or pathways. In this article, we describe the 4PD nanoplatform, which allows the in vivo preferential transfection and in vivo tracking of TEMCs with the desired RNAs. This platform is based on the conjugation of CD124/IL-4Rα-targeting peptide with G5 PAMAM dendrimers as the loading surface and can convey therapeutic or experimental RNAs of interest. When injected i.v. in mice bearing CT26 colon carcinoma or B16 melanoma, the 4PD nanoparticles predominantly accumulate at the tumor site, transfecting intratumoral myeloid cells. The use of 4PD to deliver a combination of STAT3- and C/EBPβ-specific short hairpin RNA or miR-142-3p confirmed the importance of these genes and microRNAs in TEMC biology and indicates that silencing of both genes is necessary to increase the efficacy of immune interventions. Thus, the 4PD nanoparticle can rapidly and cost effectively modulate and assess the in vivo function of microRNAs and mRNAs in TEMCs.

  18. Enhanced functional integration of human photoreceptor precursors into human and rodent retina in an ex vivo retinal explant model system.

    PubMed

    Yanai, Anat; Laver, Christopher R J; Gregory-Evans, Cheryl Y; Liu, Ran R; Gregory-Evans, Kevin

    2015-06-01

    Retinal disease is the major cause of irreversible blindness in developed countries. Transplantation of photoreceptor precursor cells (PPCs) derived from human embryonic stem cells (hESCs) is a promising and widely applicable approach for the treatment of these blinding conditions. Previously, it has been shown that after transplantation into the degenerating retina, the percentage of PPCs that undergo functional integration is low. The factors that inhibit PPC engraftment remain largely unknown, in part, because so many adverse factors could be at play during in vivo experiments. To advance our knowledge in overcoming potential adverse effects and optimize PPC transplantation, we have developed a novel ex vivo system. Harvested neural retina was placed directly on top of cultured retinal pigment epithelial (RPE) cells from a number of different sources. To mimic PPC transplantation into the subretinal space, hESC-derived PPCs were inserted between the retinal explant and underlying RPE. Explants cocultured with hESC-derived RPE maintained normal gross morphology and viability for up to 2 weeks, whereas the explants cultured on ARPE19 and RPE-J failed by 7 days. Furthermore, the proportion of PPCs expressing ribbon synapse-specific proteins BASSOON and RIBEYE was significantly higher when cocultured with hESC-derived RPE (20% and 10%, respectively), than when cocultured with ARPE19 (only 6% and 2%, respectively). In the presence of the synaptogenic factor thrombospondin-1 (TSP-1), the proportion of BASSOON-positive and RIBEYE-positive PPCs cocultured with hESC-derived RPE increased to ∼30% and 15%, respectively. These data demonstrate the utility of an ex vivo model system to define factors, such as TSP-1, which could influence integration efficiency in future in vivo experiments in models of retinal degeneration.

  19. Phenotype and functional evaluation of ex vivo generated antigen-specific immune effector cells with potential for therapeutic applications.

    PubMed

    Han, Shuhong; Huang, Yuju; Liang, Yin; Ho, Yuchin; Wang, Yichen; Chang, Lung-Ji

    2009-08-06

    Ex vivo activation and expansion of lymphocytes for adoptive cell therapy has demonstrated great success. To improve safety and therapeutic efficacy, increased antigen specificity and reduced non-specific response of the ex vivo generated immune cells are necessary. Here, using a complete protein-spanning pool of pentadecapeptides of the latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV), a weak viral antigen which is associated with EBV lymphoproliferative diseases, we investigated the phenotype and function of immune effector cells generated based on IFN-gamma or CD137 activation marker selection and dendritic cell (DC) activation. These ex vivo prepared immune cells exhibited a donor- and antigen-dependent T cell response; the IFN-gamma-selected immune cells displayed a donor-related CD4- or CD8-dominant T cell phenotype; however, the CD137-enriched cells showed an increased ratio of CD4 T cells. Importantly, the pentadecapeptide antigens accessed both class II and class I MHC antigen processing machineries and effectively activated EBV-specific CD4 and CD8 T cells. Phenotype and kinetic analyses revealed that the IFN-gamma and the CD137 selections enriched more central memory T (Tcm) cells than did the DC-activation approach, and after expansion, the IFN-gamma-selected effector cells showed the highest level of antigen-specificity and effector activities. While all three approaches generated immune cells with comparable antigen-specific activities, the IFN-gamma selection followed by ex vivo expansion produced high quality and quantity of antigen-specific effector cells. Our studies presented the optimal approach for generating therapeutic immune cells with potential for emergency and routine clinical applications.

  20. Functional organization of mitotic microtubules. Physical chemistry of the in vivo equilibrium system.

    PubMed Central

    Inoué, S; Fuseler, J; Salmon, E D; Ellis, G W

    1975-01-01

    Equilibrium between mitotic microtubules and tubulin is analyzed, using birefringence of mitotic spindle to measure microtubule concentration in vivo. A newly designed temperature-controlled slide and miniature, thermostated hydrostatic pressure chamber permit rapid alteration of temperature and of pressure. Stress birefringence of the windows is minimized, and a system for rapid recording of compensation is incorporated, so that birefringence can be measured to 0.1 nm retardation every few seconds. Both temperature and pressure data yield thermodynamic values (delta H similar to 35 kcal/mol, delta S similar to 120 entropy units [eu], delta V similar to 400 ml/mol of subunit polymerized) consistent with the explanation that polymerization of tubulin is entropy driven and mediated by hydrophobic interactions. Kinetic data suggest pseudo-zero-order polymerization and depolymerization following rapid temperature shifts, and a pseudo-first-order depolymerization during anaphase at constant temperature. The equilibrium properties of the in vivo mitotic microtubules are compared with properties of isolated brain tubules. Images FIGURE 1 FIGURE 2 FIGURE 5 FIGURE 12 FIGURE 13 FIGURE 14 FIGURE 19 PMID:1139037

  1. Application of locked nucleic acids to improve aptamer in vivo stability and targeting function

    PubMed Central

    Schmidt, Kathrin S.; Borkowski, Sandra; Kurreck, Jens; Stephens, Andrew W.; Bald, Rolf; Hecht, Maren; Friebe, Matthias; Dinkelborg, Ludger; Erdmann, Volker A.

    2004-01-01

    Aptamers are powerful candidates for molecular imaging applications due to a number of attractive features, including rapid blood clearance and tumor penetration. We carried out structure–activity relationship (SAR) studies with the Tenascin-C binding aptamer TTA1, which is a promising candidate for application in tumor imaging with radioisotopes. The aim was to improve its in vivo stability and target binding. We investigated the effect of thermal stabilization of the presumed non-binding double-stranded stem region on binding affinity and resistance against nucleolytic degradation. To achieve maximal thermal stem stabilization melting experiments with model hexanucleotide duplexes consisting of unmodified RNA, 2′-O-methyl RNA (2′-OMe), 2′-Fluoro RNA (2′-F) or Locked Nucleic Acids (LNAs) were initially carried out. Extremely high melting temperatures have been found for an LNA/LNA duplex. TTA1 derivatives with LNA and 2′-OMe modifications within the non-binding stem have subsequently been synthesized. Especially, the LNA-modified TTA1 derivative exhibited significant stem stabilization and markedly improved plasma stability while maintaining its binding affinity to the target. In addition, higher tumor uptake and longer blood retention was found in tumor-bearing nude mice. Thus, our strategy to introduce LNA modifications after the selection procedure is likely to be generally applicable to improve the in vivo stability of aptamers without compromising their binding properties. PMID:15509871

  2. Functional Analysis and Treatment of Severe Pica.

    ERIC Educational Resources Information Center

    Mace, F. Charles; Knight, David

    1986-01-01

    A two-phase functional analysis of a profoundly retarded 19-year-old male's pica behavior resulted in an effective staff-implemented treatment consisting of limited staff-client interaction and removal of a protective helmet which had previously been prescribed to help control pica. (Author/JW)

  3. In vivo Comet assay--statistical analysis and power calculations of mice testicular cells.

    PubMed

    Hansen, Merete Kjær; Sharma, Anoop Kumar; Dybdahl, Marianne; Boberg, Julie; Kulahci, Murat

    2014-11-01

    The in vivo Comet assay is a sensitive method for evaluating DNA damage. A recurrent concern is how to analyze the data appropriately and efficiently. A popular approach is to summarize the raw data into a summary statistic prior to the statistical analysis. However, consensus on which summary statistic to use has yet to be reached. Another important consideration concerns the assessment of proper sample sizes in the design of Comet assay studies. This study aims to identify a statistic suitably summarizing the % tail DNA of mice testicular samples in Comet assay studies. A second aim is to provide curves for this statistic outlining the number of animals and gels to use. The current study was based on 11 compounds administered via oral gavage in three doses to male mice: CAS no. 110-26-9, CAS no. 512-56-1, CAS no. 111873-33-7, CAS no. 79-94-7, CAS no. 115-96-8, CAS no. 598-55-0, CAS no. 636-97-5, CAS no. 85-28-9, CAS no. 13674-87-8, CAS no. 43100-38-5 and CAS no. 60965-26-6. Testicular cells were examined using the alkaline version of the Comet assay and the DNA damage was quantified as % tail DNA using a fully automatic scoring system. From the raw data 23 summary statistics were examined. A linear mixed-effects model was fitted to the summarized data and the estimated variance components were used to generate power curves as a function of sample size. The statistic that most appropriately summarized the within-sample distributions was the median of the log-transformed data, as it most consistently conformed to the assumptions of the statistical model. Power curves for 1.5-, 2-, and 2.5-fold changes of the highest dose group compared to the control group when 50 and 100 cells were scored per gel are provided to aid in the design of future Comet assay studies on testicular cells.

  4. The functional analysis of problematic verbal behavior

    PubMed Central

    Ewing, Christopher B.; Magee, Sandy K.; Ellis, Janet

    2002-01-01

    This study describes procedures and outcomes in a functional analysis of problem behavior of 2 public school students. For a 13-year-old honors student, bizarre tacts (labeled as psychotic speech by school staff) were maintained by attention. For a 15-year-old with autism, the functional analysis revealed that perseverative mands for toileting were controlled by attention; mands for edible items were controlled by access to any food item; and mands for nonedible items were maintained by access to the specific item manded. The “problematic” aspects of the verbal behavior differed—the bizarre speech was problematic based on its content, but the perseverative verbalizations resulted in high response cost for classroom staff. Research in the area of problematic verbal behavior is sparse and warrants further attention from behavior analysts who work in public school settings. This research demonstrates the applicability and relevance of functionally analyzing problematic verbal behavior in public school settings. PMID:22477228

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

    PubMed

    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.

  6. Improving information retrieval in functional analysis.

    PubMed

    Rodriguez, Juan C; González, Germán A; Fresno, Cristóbal; Llera, Andrea S; Fernández, Elmer A

    2016-12-01

    Transcriptome analysis is essential to understand the mechanisms regulating key biological processes and functions. The first step usually consists of identifying candidate genes; to find out which pathways are affected by those genes, however, functional analysis (FA) is mandatory. The most frequently used strategies for this purpose are Gene Set and Singular Enrichment Analysis (GSEA and SEA) over Gene Ontology. Several statistical methods have been developed and compared in terms of computational efficiency and/or statistical appropriateness. However, whether their results are similar or complementary, the sensitivity to parameter settings, or possible bias in the analyzed terms has not been addressed so far. Here, two GSEA and four SEA methods and their parameter combinations were evaluated in six datasets by comparing two breast cancer subtypes with well-known differences in genetic background and patient outcomes. We show that GSEA and SEA lead to different results depending on the chosen statistic, model and/or parameters. Both approaches provide complementary results from a biological perspective. Hence, an Integrative Functional Analysis (IFA) tool is proposed to improve information retrieval in FA. It provides a common gene expression analytic framework that grants a comprehensive and coherent analysis. Only a minimal user parameter setting is required, since the best SEA/GSEA alternatives are integrated. IFA utility was demonstrated by evaluating four prostate cancer and the TCGA breast cancer microarray datasets, which showed its biological generalization capabilities.

  7. In vivo ultrasound assessment of respiratory function of abdominal muscles in normal subjects.

    PubMed

    Misuri, G; Colagrande, S; Gorini, M; Iandelli, I; Mancini, M; Duranti, R; Scano, G

    1997-12-01

    Ultrasonography has recently been proposed for assessing changes in thickness and motion of the diaphragm during contraction in humans. Data on ultrasound assessment of abdominal muscles in humans are scarce. We therefore investigated the changes in thickness and the relevant mechanical effects of abdominal muscles using this technique during respiratory manoeuvres in normal subjects. We evaluated the thickness of the abdominal muscle layers in six normal male subjects (aged 26-36 yrs) using a 7.5 MHz B-mode ultrasound transducer. Gastric (Pg) and mouth pressures, muscle thickness of external oblique (EO), internal oblique (IO), transversus abdominis (TA) and rectus abdominis (RA) were assessed at functional residual capacity (FRC), residual volume (RV), total lung capacity (TLC), during progressive (PEEs) and maximal expiratory efforts (MEEs) against a closed airway and during homolateral (HTR) and contralateral (CTR) trunk rotation. Abdominal muscle thickness was found to be reproducible (coefficient of variation and two-way analysis of variance). Compared to FRC, the thickness of IO, TA and RA significantly increased at RV and during MEEs, whereas EO remained unchanged; at TLC, the thickness of IO and TA significantly decreased. During PEEs, a significant relationship between increase in Pg and TA thickness was observed in all subjects, the thickness of the other abdominal muscles being inconsistently related to Pg. Finally, a significant increase in the thickness of IO and EO was found during HTR and CTR, respectively. We conclude that during maximal expiratory manoeuvres, transversus abdominis, internal oblique and rectus abdominis thickened similarly. Transversus abdominis seems to be the major contributor in generating abdominal expiratory pressure during progressive expiratory efforts. External oblique seems to be preferentially involved during trunk rotation. These results suggest the possible value of studying the abdominal muscles by ultrasonography in

  8. Functional analysis and treatment of elopement.

    PubMed Central

    Piazza, C C; Hanley, G P; Bowman, L G; Ruyter, J M; Lindauer, S E; Saiontz, D M

    1997-01-01

    Elopement is a dangerous behavior because children who run away may encounter life-threatening situations (e.g., traffic). We conducted functional analyses of the elopement of 3 children who had been diagnosed with developmental disabilities. The results identified a maintaining reinforcer for the elopement of 1 child, but the data were difficult to interpret for 2 of the children. Subsequent reinforcer assessments were used to help to clarify the reinforcers for elopement for these 2 children. Results of the functional analyses and reinforcer assessments then were used to develop successful treatments to reduce elopement. The findings are discussed in terms of (a) the application of functional analysis methodology to elopement, (b) the use of reinforcer assessments to identify potential reinforcers when standard functional analyses are undifferentiated, and (c) the utility of assessment-based treatments for elopement. PMID:9433790

  9. Kupfer-type immunological synapse characteristics do not predict anti-brain tumor cytolytic T-cell function in vivo.

    PubMed

    Yang, J; Sanderson, N S R; Wawrowsky, K; Puntel, M; Castro, M G; Lowenstein, P R

    2010-03-09

    To analyze the in vivo structure of antigen-specific immunological synapses during an effective immune response, we established brain tumors expressing the surrogate tumor antigen ovalbumin and labeled antigen-specific anti-glioma T cells using specific tetramers. Using these techniques, we determined that a significant number of antigen-specific T cells were localized to the brain tumor and surrounding brain tissue and a large percentage could be induced to express IFNgamma when exposed to the specific ovalbumin-derived peptide epitope SIINFEKL. Detailed morphological analysis of T cells immunoreactive for tetramers in direct physical contact with tumor cells expressing ovalbumin indicated that the interface between T cells and target tumor cells displayed various morphologies, including Kupfer-type immunological synapses. Quantitative analysis of adjacent confocal optical sections was performed to determine if the higher frequency of antigen-specific antiglioma T cells present in animals that developed an effective antitumor immune response could be correlated with a specific immunological synaptic morphology. Detailed in vivo quantitative analysis failed to detect an increased proportion of immunological synapses displaying the characteristic Kupfer-type morphology in animals mounting a strong and effective antitumor immune response as compared with those experiencing a clinically ineffective response. We conclude that an effective cytolytic immune response is not dependent on an increased frequency of Kupfer-type immunological synapses between T cells and tumor cells.

  10. Slam haplotypes modulate the response to LPS in vivo through control of NKT cell number and function1

    PubMed Central

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

    2011-01-01

    CD1d-restricted NKT cells comprise an innate-like T cell subset that hasbeen demonstrated to play a role in amplifying the response of innate immune leukocytesto TLR ligands. The Slam locus contains genes that have been implicated in both innate and adaptive immune responses. Here, we demonstrate that divergent Slam locus haplotypesmodulate the response of macrophages to TLR ligands such as LPS through their control of NKT cell number and function. In response to LPS challenge in vivo, macrophage TNF production in Slam haplotype-2-associated 129S1/SvImJ and 129X1/SvJ mice was significantly impaired in comparison to macrophage TNF production in Slam haplotype -1-positive 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 α-galactosylceramide, and in NKT cell IFN-γ production after LPS challenge in vivo. Using B6.129 c1congenic mice and adoptive transfer, we found that divergent Slam haplotypes controlled both 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 SLAM family receptors on NKT cells. These data suggest that the polymorphisms that distinguish two Slam haplotypes significantly modulate the innate immune response in vivothrough their effect on NKT cell s. PMID:20530260

  11. A comparison of InVivoStat with other statistical software packages for analysis of data generated from animal experiments.

    PubMed

    Clark, Robin A; Shoaib, Mohammed; Hewitt, Katherine N; Stanford, S Clare; Bate, Simon T

    2012-08-01

    InVivoStat is a free-to-use statistical software package for analysis of data generated from animal experiments. The package is designed specifically for researchers in the behavioural sciences, where exploiting the experimental design is crucial for reliable statistical analyses. This paper compares the analysis of three experiments conducted using InVivoStat with other widely used statistical packages: SPSS (V19), PRISM (V5), UniStat (V5.6) and Statistica (V9). We show that InVivoStat provides results that are similar to those from the other packages and, in some cases, are more advanced. This investigation provides evidence of further validation of InVivoStat and should strengthen users' confidence in this new software package.

  12. Effect of antimalarials treatment on rat liver lysosomal function-Anin vivo study.

    PubMed

    Patel, Samir P; Katewa, Subhash D; Katyare, Surendra S

    2005-01-01

    Effects of treatmentin vivo with the antimalarials:chloroquine (CQ), primaquine (PQ) and quinine(Q) on lysosomal enzymes and lysosomal membrane integrity were examined. Treatment with the three antimalarials showed an apparent increase in the membrane stability. CQ treatment resulted in increase in both the 'free' and 'total' activities of all the enzymes i.e. acid phosphatase, RNase II, DNase II and cathepsin D. PQ treatment lowered the 'free' and 'total' activities of acid phosphatase and cathepsin D, but the DNase II activities increased. Treatment with Q resulted in increased 'free' and 'total' activities of RNase II and DNase II. While 'free' activities of acid phosphatase and cathepsin D were low; the 'total' activities increased significantly. Our results suggest that a generalized increase in free nucleases activities following prolonged treatment with antimalarials may lead to cell damage and/or necrosis.

  13. Gold and Hairpin DNA Functionalization of Upconversion Nanocrystals for Imaging and In Vivo Drug Delivery.

    PubMed

    Han, Sanyang; Samanta, Animesh; Xie, Xiaoji; Huang, Ling; Peng, Juanjuan; Park, Sung Jin; Teh, Daniel Boon Loong; Choi, Yongdoo; Chang, Young-Tae; All, Angelo Homayoun; Yang, Yanmei; Xing, Bengang; Liu, Xiaogang

    2017-03-10

    Although multifunctional upconversion imaging probes have recently attracted considerable interest in biomedical research, there are currently few methods for stabilizing these luminescent nanoprobes with oligonucleotides in biological systems. Herein, a method to robustly disperse upconversion nanoprobes in physiological buffers based on rational design and synthesis of nanoconjugates comprising hairpin-DNA-modified gold nanoparticles is presented. This approach imparts the upconversion nanoprobes with excellent biocompatibility and circumvents the problem of particle agglomeration. By combining single-band anti-Stokes near-infrared emission and the photothermal effect mediated by the coupling of gold to upconversion nanoparticles, a simple, versatile nanoparticulate system for simultaneous deep-tissue imaging and drug molecule release in vivo is demonstrated.

  14. In vivo function of immune murine peritoneal exudate cells after freezing and thawing

    SciTech Connect

    Adkison, L.R.; Coggin, J.H. Jr.

    1980-01-01

    Peritoneal exudate cells were collected from Balb/c mice immunized against a 3-methylcholanthrene-induced (3-MCA) tumor and known to be capable of conferring tumor transplantation resistance in vivo in syngeneic recipients. These PEC were frozen-using dimethylsulfoxide as the cryopreservative agent. Adoptive transfer of tumor resistance in syngeneic recipients challenged with homologous 3-MCA sarcoma cells was attempted using these frozen exudate cells. Cells were thawed 1, 4, 7, 10 or 30 days after freezing and admixed with tumor cells in ratios of 100:1 or 1000:1 before injecting into mice. Tumorigenesis was decreased and delayed in groups receiving the 100:1 ratio. Less than 3% of the mice developed tumors in groups receiving the 1000:1 ratio. The number of cells recovered post-thawing ranged from 60 to 80%; viability of post-thawed cells ranged from 80 to 96%.

  15. Genetic analysis of glutamatergic function in Drosophila

    SciTech Connect

    Chase, B.A.; Kankel, D.R.

    1987-01-01

    Neurotransmitters are essential for communication between neurons and hence are vital in the overall integrative functioning of the nervous system. Previous work on acetylcholine metabolism in the fruit fly, Drosophila melanogaster, has also raised the possibility that transmitter metabolism may play a prominent role in either the achievement or maintenance of the normal structure of the central nervous system in this species. Unfortunately, acetylcholine is rather poorly characterized as a neurotransmitter in Drosophila; consequently, we have begun an analysis of the role of glutamate (probably the best characterized transmitter in this organism) in the formation and/or maintenance of nervous system structure. We present here the results of a series of preliminary analyses. To suggest where glutamatergic function may be localized, an examination of the spatial distribution of high affinity (/sup 3/H)-glutamate binding sites are presented. We present the results of an analysis of the spatial and temporal distribution of enzymatic activities thought to be important in the regulation of transmitter-glutamate pools (i.e., glutamate oxaloacetic transaminase, glutaminase, and glutamate dehydrogenase). To begin to examine whether mutations in any of these functions are capable of affecting glutamatergic activity, we present the results of an initial genetic analysis of one enzymatic function, glutamate oxaloacetic transaminase (GOT), chosen because of its differential distribution within the adult central nervous system and musculature.

  16. Cocaine- and amphetamine-regulated transcript (CART) peptide as an in vivo regulator of cardiac function in Rana ridibunda frog.

    PubMed

    Ivanova, Iliyana V; Schubert, Rudolf; Duridanova, Dessislava B; Bolton, Thomas B; Lubomirov, Lubomir T; Gagov, Hristo S

    2007-11-01

    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. The CART peptide, when injected into the venous sinus, significantly and reproducibly increased the force of frog heart contractions by up to 33.0 +/- 6.4% during the first 15 min after its application but did not influence the chronotropic activity of the frog heart. The positive inotropic effect was entirely blocked by prazosin, pertussis toxin, R(p)-adenosine 3',5'-cyclic monophosphorothioate, autosauvagine 30 or metyrapone, as well as by extirpation of the pituitary gland, functional elimination of the inter-renal glands and long-lasting starvation, and was not observed on isolated heart preparations. Propranolol and double pithing were without significant effect on this phenomenon. It was concluded that: (i) CART peptide, administered to frogs in vivo, increases the force of heart contractions; (ii) this effect of the peptide is exerted via activation of the hypothalamic-pituitary-inter-renal gland axis through a corticoliberin-sensitive mechanism; (iii) CART augments the pumping function of the heart via a corticosteroid-dependent potentiation of myocardial alpha(1)-adrenoreceptors signalling; and (iv) prolonged food deprivation abolishes the positive inotropic effect of CART, suggesting the participation of endogenous CART in the physiological adaptation of the circulatory system to limitations of energy consumption.

  17. Tartary buckwheat improves cognition and memory function in an in vivo amyloid-β-induced Alzheimer model.

    PubMed

    Choi, Ji Yeon; Cho, Eun Ju; Lee, Hae Song; Lee, Jeong Min; Yoon, Young-Ho; Lee, Sanghyun

    2013-03-01

    Protective effects of Tartary buckwheat (TB) and common buckwheat (CB) on amyloid beta (Aβ)-induced impairment of cognition and memory function were investigated in vivo in order to identify potential therapeutic agents against Alzheimer's disease (AD) and its associated progressive memory deficits, cognitive impairment, and personality changes. An in vivo mouse model of AD was created by injecting the brains of ICR mice with Aβ(25-35), a fragment of the full-length Aβ protein. Damage of mice recognition ability through following Aβ(25-35) brain injections was confirmed using the T-maze test, the object recognition test, and the Morris water maze test. Results of behavior tests in AD model showed that oral administration of the methanol (MeOH) extracts of TB and CB improved cognition and memory function following Aβ(25-35) injections. Furthermore, in groups receiving the MeOH extracts of TB and CB, lipid peroxidation was significantly inhibited, and nitric oxide levels in tissue, which are elevated by injection of Aβ(25-35), were also decrease. In particular, the MeOH extract of TB exerted a stronger protective activity than CB against Aβ(25-35)-induced memory and cognition impairment. The results indicate that TB may play a promising role in preventing or reversing memory and cognition loss associated with Aβ(25-35)-induced AD.

  18. The rare DAT coding variant Val559 perturbs DA neuron function, changes behavior, and alters in vivo responses to psychostimulants.

    PubMed

    Mergy, Marc A; Gowrishankar, Raajaram; Gresch, Paul J; Gantz, Stephanie C; Williams, John; Davis, Gwynne L; Wheeler, C Austin; Stanwood, Gregg D; Hahn, Maureen K; Blakely, Randy D

    2014-11-04

    Despite the critical role of the presynaptic dopamine (DA) transporter (DAT, SLC6A3) in DA clearance and psychostimulant responses, evidence that DAT dysfunction supports risk for mental illness is indirect. Recently, we identified a rare, nonsynonymous Slc6a3 variant that produces the DAT substitution Ala559Val in two male siblings who share a diagnosis of attention-deficit hyperactivity disorder (ADHD), with other studies identifying the variant in subjects with bipolar disorder (BPD) and autism spectrum disorder (ASD). Previously, using transfected cell studies, we observed that although DAT Val559 displays normal total and surface DAT protein levels, and normal DA recognition and uptake, the variant transporter exhibits anomalous DA efflux (ADE) and lacks capacity for amphetamine (AMPH)-stimulated DA release. To pursue the significance of these findings in vivo, we engineered DAT Val559 knock-in mice, and here we demonstrate in this model the presence of elevated extracellular DA levels, altered somatodendritic and presynaptic D2 DA receptor (D2R) function, a blunted ability of DA terminals to support depolarization and AMPH-evoked DA release, and disruptions in basal and psychostimulant-evoked locomotor behavior. Together, our studies demonstrate an in vivo functional impact of the DAT Val559 variant, providing support for the ability of DAT dysfunction to impact risk for mental illness.

  19. Mammary extracellular matrix directs differentiation of testicular and embryonic stem cells to form functional mammary glands in vivo

    PubMed Central

    Bruno, Robert D.; Fleming, Jodie M.; George, Andrea L.; Boulanger, Corinne A.; Schedin, Pepper; Smith, Gilbert H.

    2017-01-01

    Previously, we demonstrated the ability of the normal mammary microenvironment (niche) to direct non-mammary cells including testicular and embryonic stem cells (ESCs) to adopt a mammary epithelial cell (MEC) fate. These studies relied upon the interaction of transplanted normal MECs with non-mammary cells within the mammary fat-pads of recipient mice that had their endogenous epithelium removed. Here, we tested whether acellular mammary extracellular matrix (mECM) preparations are sufficient to direct differentiation of testicular-derived cells and ESCs to form functional mammary epithelial trees in vivo. We found that mECMs isolated from adult mice and rats were sufficient to redirect testicular derived cells to produce normal mammary epithelial trees within epithelial divested mouse mammary fat-pads. Conversely, ECMs isolated from omental fat and lung did not redirect testicular cells to a MEC fate, indicating the necessity of tissue specific components of the mECM. mECM preparations also completely inhibited teratoma formation from ESC inoculations. Further, a phenotypically normal ductal outgrowth resulted from a single inoculation of ESCs and mECM. To the best of our knowledge, this is the first demonstration of a tissue specific ECM driving differentiation of cells to form a functional tissue in vivo. PMID:28071703

  20. Incipient Social Groups: An Analysis via In-Vivo Behavioral Tracking

    PubMed Central

    Halberstadt, Jamin; Jackson, Joshua Conrad; Bilkey, David; Jong, Jonathan; Whitehouse, Harvey; McNaughton, Craig; Zollmann, Stefanie

    2016-01-01

    Social psychology is fundamentally the study of individuals in groups, yet there remain basic unanswered questions about group formation, structure, and change. We argue that the problem is methodological. Until recently, there was no way to track who was interacting with whom with anything approximating valid resolution and scale. In the current study we describe a new method that applies recent advances in image-based tracking to study incipient group formation and evolution with experimental precision and control. In this method, which we term “in vivo behavioral tracking,” we track individuals’ movements with a high definition video camera mounted atop a large field laboratory. We report results of an initial study that quantifies the composition, structure, and size of the incipient groups. We also apply in-vivo spatial tracking to study participants’ tendency to cooperate as a function of their embeddedness in those crowds. We find that participants form groups of seven on average, are more likely to approach others of similar attractiveness and (to a lesser extent) gender, and that participants’ gender and attractiveness are both associated with their proximity to the spatial center of groups (such that women and attractive individuals are more likely than men and unattractive individuals to end up in the center of their groups). Furthermore, participants’ proximity to others early in the study predicted the effort they exerted in a subsequent cooperative task, suggesting that submergence in a crowd may predict social loafing. We conclude that in vivo behavioral tracking is a uniquely powerful new tool for answering longstanding, fundamental questions about group dynamics. PMID:27007952

  1. Insulin-Producing Endocrine Cells Differentiated In Vitro From Human Embryonic Stem Cells Function in Macroencapsulation Devices In Vivo

    PubMed Central

    Ambruzs, Dana M.; Moorman, Mark A.; Bhoumik, Anindita; Cesario, Rosemary M.; Payne, Janice K.; Kelly, Jonathan R.; Haakmeester, Carl; Srijemac, Robert; Wilson, Alistair Z.; Kerr, Justin; Frazier, Mauro A.; Kroon, Evert J.; D’Amour, Kevin A.

    2015-01-01

    The PEC-01 cell population, differentiated from human embryonic stem cells (hESCs), contains pancreatic progenitors (PPs) that, when loaded into macroencapsulation devices (to produce the VC-01 candidate product) and transplanted into mice, can mature into glucose-responsive insulin-secreting cells and other pancreatic endocrine cells involved in glucose metabolism. We modified the protocol for making PEC-01 cells such that 73%–80% of the cell population consisted of PDX1-positive (PDX1+) and NKX6.1+ PPs. The PPs were further differentiated to islet-like cells (ICs) that reproducibly contained 73%–89% endocrine cells, of which approximately 40%–50% expressed insulin. A large fraction of these insulin-positive cells were single hormone-positive and expressed the transcription factors PDX1 and NKX6.1. To preclude a significant contribution of progenitors to the in vivo function of ICs, we used a simple enrichment process to remove remaining PPs, yielding aggregates that contained 93%–98% endocrine cells and 1%–3% progenitors. Enriched ICs, when encapsulated and implanted into mice, functioned similarly to the VC-01 candidate product, demonstrating conclusively that in vitro-produced hESC-derived insulin-producing cells can mature and function in vivo in devices. A scaled version of our suspension culture was used, and the endocrine aggregates could be cryopreserved and retain functionality. Although ICs expressed multiple important β cell genes, the cells contained relatively low levels of several maturity-associated markers. Correlating with this, the time to function of ICs was similar to PEC-01 cells, indicating that ICs required cell-autonomous maturation after delivery in vivo, which would occur concurrently with graft integration into the host. Significance Type 1 diabetes (T1D) affects approximately 1.25 million people in the U.S. alone and is deadly if not managed with insulin injections. This paper describes the production of insulin

  2. High resolution Physio-chemical Tissue Analysis: Towards Non-invasive In Vivo Biopsy

    PubMed Central

    Xu, Guan; Meng, Zhuo-xian; Lin, Jian-die; Deng, Cheri X.; Carson, Paul L.; Fowlkes, J. Brian; Tao, Chao; Liu, Xiaojun; Wang, Xueding

    2016-01-01

    Conventional gold standard histopathologic diagnosis requires information of both high resolution structural and chemical changes in tissue. Providing optical information at ultrasonic resolution, photoacoustic (PA) technique could provide highly sensitive and highly accurate tissue characterization noninvasively in the authentic in vivo environment, offering a replacement for histopathology. A two-dimensional (2D) physio-chemical spectrogram (PCS) combining micrometer to centimeter morphology and chemical composition simultaneously can be generated for each biological sample with PA measurements at multiple optical wavelengths. This spectrogram presents a unique 2D “physio-chemical signature” for any specific type of tissue. Comprehensive analysis of PCS, termed PA physio-chemical analysis (PAPCA), can lead to very rich diagnostic information, including the contents of all relevant molecular and chemical components along with their corresponding histological microfeatures, comparable to those accessible by conventional histology. PAPCA could contribute to the diagnosis of many diseases involving diffusive patterns such as fatty liver. PMID:26842459

  3. High resolution Physio-chemical Tissue Analysis: Towards Non-invasive In Vivo Biopsy

    NASA Astrophysics Data System (ADS)

    Xu, Guan; Meng, Zhuo-Xian; Lin, Jian-Die; Deng, Cheri X.; Carson, Paul L.; Fowlkes, J. Brian; Tao, Chao; Liu, Xiaojun; Wang, Xueding

    2016-02-01

    Conventional gold standard histopathologic diagnosis requires information of both high resolution structural and chemical changes in tissue. Providing optical information at ultrasonic resolution, photoacoustic (PA) technique could provide highly sensitive and highly accurate tissue characterization noninvasively in the authentic in vivo environment, offering a replacement for histopathology. A two-dimensional (2D) physio-chemical spectrogram (PCS) combining micrometer to centimeter morphology and chemical composition simultaneously can be generated for each biological sample with PA measurements at multiple optical wavelengths. This spectrogram presents a unique 2D “physio-chemical signature” for any specific type of tissue. Comprehensive analysis of PCS, termed PA physio-chemical analysis (PAPCA), can lead to very rich diagnostic information, including the contents of all relevant molecular and chemical components along with their corresponding histological microfeatures, comparable to those accessible by conventional histology. PAPCA could contribute to the diagnosis of many diseases involving diffusive patterns such as fatty liver.

  4. Epigenetic modulation of human breast cancer by metallofullerenol nanoparticles: in vivo treatment and in vitro analysis

    NASA Astrophysics Data System (ADS)

    Meng, Jie; Xing, Jianmin; Wang, Yingze; Lu, Juan; Zhao, Yuliang; Gao, Xueyun; Wang, Paul C.; Jia, Lee; Liang, Xingjie

    2011-11-01

    Multi-hydroxylated endohedral metallofullerenol [Gd@C82(OH)22]n nanoparticles possess the general physico-chemical characteristics of most nanoparticles. They also exhibit uniquely low toxicity and antineoplastic efficacy. In the current study, the molecular mechanisms and epigenetic characteristics of the antineoplastic action of these nanoparticles are explored. Human breast cancer MCF-7 and human umbilical vein endothelial ECV304 cell lines were used. Cell viability assay, cell hierarchical cluster analysis by cDNA microarray, semi-quantitative reverse transcription-polymerase chain reaction and Western blot analysis were conducted to investigate the changes in molecular and cellular signaling pathways caused by [Gd@C82(OH)22]n. The results demonstrated the high antitumor activity and low cytotoxicity of [Gd@C82(OH)22]n nanoparticles both in vivo and in vitro. Their possible anti-tumor mechanisms were also discussed. The present study may provide new insight into the mechanism of action of these nanoparticles.

  5. Predicting Drug Response in Human Prostate Cancer from Preclinical Analysis of In Vivo Mouse Models.

    PubMed

    Mitrofanova, Antonina; Aytes, Alvaro; Zou, Min; Shen, Michael M; Abate-Shen, Cory; Califano, Andrea

    2015-09-29

    Although genetically engineered mouse (GEM) models are often used to evaluate cancer therapies, extrapolation of such preclinical data to human cancer can be challenging. Here, we introduce an approach that uses drug perturbation data from GEM models to predict drug efficacy in human cancer. Network-based analysis of expression profiles from in vivo treatment of GEM models identified drugs and drug combinations that inhibit the activity of FOXM1 and CENPF, which are master regulators of prostate cancer malignancy. Validation of mouse and human prostate cancer models confirmed the specificity and synergy of a predicted drug combination to abrogate FOXM1/CENPF activity and inhibit tumorigenicity. Network-based analysis of treatment signatures from GEM models identified treatment-responsive genes in human prostate cancer that are potential biomarkers of patient response. More generally, this approach allows systematic identification of drugs that inhibit tumor dependencies, thereby improving the utility of GEM models for prioritizing drugs for clinical evaluation.

  6. Automated in vivo 3D high-definition optical coherence tomography skin analysis system.

    PubMed

    Ai Ping Yow; Jun Cheng; Annan Li; Srivastava, Ruchir; Jiang Liu; Wong, Damon Wing Kee; Hong Liang Tey

    2016-08-01

    The in vivo assessment and visualization of skin structures can be performed through the use of high resolution optical coherence tomography imaging, also known as HD-OCT. However, the manual assessment of such images can be exhaustive and time consuming. In this paper, we present an analysis system to automatically identify and quantify the skin characteristics such as the topography of the surface of the skin and thickness of the epidermis in HD-OCT images. Comparison of this system with manual clinical measurements demonstrated its potential for automatic objective skin analysis and diseases diagnosis. To our knowledge, this is the first report of an automated system to process and analyse HD-OCT skin images.

  7. CD22 regulates B lymphocyte function in vivo through both ligand-dependent and ligand-independent mechanisms.

    PubMed

    Poe, Jonathan C; Fujimoto, Yoko; Hasegawa, Minoru; Haas, Karen M; Miller, Ann S; Sanford, Isaac G; Bock, Cheryl B; Fujimoto, Manabu; Tedder, Thomas F

    2004-10-01

    The interaction of CD22 with alpha2,6-linked sialic acid ligands has been widely proposed to regulate B lymphocyte function and migration. Here, we generated gene-targeted mice that express mutant CD22 molecules that do not interact with these ligands. CD22 ligand binding regulated the expression of cell surface CD22, immunoglobulin M and major histocompatibility complex class II on mature B cells, maintenance of the marginal zone B cell population, optimal B cell antigen receptor-induced proliferation, and B cell turnover rates. However, CD22 negative regulation of calcium mobilization after B cell antigen receptor ligation, CD22 phosphorylation, recruitment of SHP-1 to CD22 and B cell migration did not require CD22 ligand engagement. These observations resolve longstanding questions regarding the physiological importance of CD22 ligand binding in the regulation of B cell function in vivo.

  8. Monofrequency forced oscillation technique for the investigation of pulmonary function in calves: in vitro and in vivo studies.

    PubMed

    Close, R; Reinhold, P; Lekeux, P

    1994-05-01

    Parameters derived from the non-invasive and simple monofrequency forced oscillation technique were compared with classical parameters of ventilatory mechanics in order to assess its usefulness for the investigation of pulmonary function in calves. To facilitate this comparison, theoretical derivations were coupled with in vitro measurements, using an artificial lung model, and with in vivo studies. These studies compared the oscillatory resistance parameters (Ros and Re) and the respiratory system compliance (Crs) against the classical pulmonary resistance (RL) and the dynamic compliance (Cdyn), respectively. Ros and Re were highly correlated (r > or = 0.87) with RL and the comparison between Crs and Cdyn gave a similarly high correlation (r > or = 0.88). Given its simplicity, its correspondence with classical parameters and its rapidity and reproducibility, monofrequency forced oscillation technique seems well suited for the investigation of pulmonary function under field conditions.

  9. SMJ's analysis of Ising model correlation functions

    NASA Astrophysics Data System (ADS)

    Kadanoff, Leo P.; Kohmoto, Mahito

    1980-05-01

    In a series of recent publications Sato, Miwa, and Jimbo (SMJ) have shown how to derive multispin correlation functions of the two-dimensional Ising model in the continuum, or scaling, limit by analyzing the behavior of the solutions to the two-dimensional version of the Dirac equation. The major purpose of the present work is to describe SMJ's analysis more discursively and in terms closer to that used in previous studies of the Ising model. In addition, new and more compact expressions for their basic equations are derived. A single new answer is obtained: the form of the three-spin correlation function at criticality.

  10. Dispersion analysis with inverse dielectric function modelling.

    PubMed

    Mayerhöfer, Thomas G; Ivanovski, Vladimir; Popp, Jürgen

    2016-11-05

    We investigate how dispersion analysis can profit from the use of a Lorentz-type description of the inverse dielectric function. In particular at higher angles of incidence, reflectance spectra using p-polarized light are dominated by bands from modes that have their transition moments perpendicular to the surface. Accordingly, the spectra increasingly resemble inverse dielectric functions. A corresponding description can therefore eliminate the complex dependencies of the dispersion parameters, allow their determination and facilitate a more accurate description of the optical properties of single crystals.

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

    PubMed

    Wine, Jeffrey J; Char, Jessica E; Chen, Jonathan; Cho, Hyung-Ju; Dunn, Colleen; Frisbee, Eric; Joo, Nam Soo; Milla, Carlos; Modlin, Sara E; Park, Il-Ho; Thomas, Ewart A C; Tran, Kim V; Verma, Rohan; Wolfe, Marlene H

    2013-01-01

    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.

  12. Lipopolysaccharide enhances FcγR-dependent functions in vivo through CD11b/CD18 up-regulation

    PubMed Central

    Rubel, C; Miliani De Marval, P; Vermeulen, M; Isturiz, M A; Palermo, M S

    1999-01-01

    Fc receptors for immunoglobulin G (IgG) (FcγR) mediate several defence mechanisms in the course of inflammatory and infectious diseases. In Gram-negative infections, cellular wall lipopolysaccharides (LPS) modulate different immune responses. We have recently demonstrated that murine LPS in vivo treatment significantly increases FcγR-dependent clearance of immune complexes (IC). In addition, we and others have reported the induction of adhesion molecules on macrophages and neutrophils by LPS in vivo and by tumour necrosis factor-α (TNF-α) in vitro. The aim of this paper was to investigate CD11b/CD18 participation in LPS enhancing effects on Fcγ-dependent functionality of tissue macrophages. Our results have demonstrated that LPS can enhance antibody-dependent cellular cytotoxicity (ADCC) and IC-triggered cytotoxicity (IC-Ctx), two reactions which involve the Fcγ-receptor but different lytic mechanisms. In vitro incubation of splenocytes from LPS-treated mice with anti-CD11b/CD18 abrogated ADCC and IC-Ctx enhancement, without affecting FcγR expression. Similar results were obtained with physiological concentrations of fibrinogen. In this way cytotoxic values of LPS-splenocytes decreased to the basal levels of control mice. Time and temperature requirements for such inhibition strongly suggested that anti-CD11b/CD18 could modulate intracellular signals leading to downregulation of FcγR functionality. Data presented herein support the hypothesis that functional and/or physical associations between integrins and FcγR could be critical for the modulation of effector functions during an inflammatory response. PMID:10447764

  13. Functional Data Analysis of Tree Data Objects.

    PubMed

    Shen, Dan; Shen, Haipeng; Bhamidi, Shankar; Maldonado, Yolanda Muñoz; Kim, Yongdai; Marron, J S

    2014-01-01

    Data analysis on non-Euclidean spaces, such as tree spaces, can be challenging. The main contribution of this paper is establishment of a connection between tree data spaces and the well developed area of Functional Data Analysis (FDA), where the data objects are curves. This connection comes through two tree representation approaches, the Dyck path representation and the branch length representation. These representations of trees in Euclidean spaces enable us to exploit the power of FDA to explore statistical properties of tree data objects. A major challenge in the analysis is the sparsity of tree branches in a sample of trees. We overcome this issue by using a tree pruning technique that focuses the analysis on important underlying population structures. This method parallels scale-space analysis in the sense that it reveals statistical properties of tree structured data over a range of scales. The effectiveness of these new approaches is demonstrated by some novel results obtained in the analysis of brain artery trees. The scale space analysis reveals a deeper relationship between structure and age. These methods are the first to find a statistically significant gender difference.

  14. Biosensors for functional food safety and analysis.

    PubMed

    Lavecchia, Teresa; Tibuzzi, Arianna; Giardi, Maria Teresa

    2010-01-01

    The importance of safety and functionality analysis of foodstuffs and raw materials is supported by national legislations and European Union (EU) directives concerning not only the amount of residues of pollutants and pathogens but also the activity and content of food additives and the health claims stated on their labels. In addition, consumers' awareness of the impact of functional foods' on their well-being and their desire for daily healthcare without the intake pharmaceuticals has immensely in recent years. Within this picture, the availability of fast, reliable, low cost control systems to measure the content and the quality of food additives and nutrients with health claims becomes mandatory, to be used by producers, consumers and the governmental bodies in charge of the legal supervision of such matters. This review aims at describing the most important methods and tools used for food analysis, starting with the classical methods (e.g., gas-chromatography GC, high performance liquid chromatography HPLC) and moving to the use of biosensors-novel biological material-based equipments. Four types of bio-sensors, among others, the novel photosynthetic proteins-based devices which are more promising and common in food analysis applications, are reviewed. A particular highlight on biosensors for the emerging market of functional foods is given and the most widely applied functional components are reviewed with a comprehensive analysis of papers published in the last three years; this report discusses recent trends for sensitive, fast, repeatable and cheap measurements, focused on the detection of vitamins, folate (folic acid), zinc (Zn), iron (Fe), calcium (Ca), fatty acids (in particular Omega 3), phytosterols and phytochemicals. A final market overview emphasizes some practical aspects ofbiosensor applications.

  15. Statistical strategies to reveal potential vibrational markers for in vivo analysis by confocal Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Oliveira Mendes, Thiago de; Pinto, Liliane Pereira; Santos, Laurita dos; Tippavajhala, Vamshi Krishna; Téllez Soto, Claudio Alberto; Martin, Airton Abrahão

    2016-07-01

    The analysis of biological systems by spectroscopic techniques involves the evaluation of hundreds to thousands of variables. Hence, different statistical approaches are used to elucidate regions that discriminate classes of samples and to propose new vibrational markers for explaining various phenomena like disease monitoring, mechanisms of action of drugs, food, and so on. However, the technical statistics are not always widely discussed in applied sciences. In this context, this work presents a detailed discussion including the various steps necessary for proper statistical analysis. It includes univariate parametric and nonparametric tests, as well as multivariate unsupervised and supervised approaches. The main objective of this study is to promote proper understanding of the application of various statistical tools in these spectroscopic methods used for the analysis of biological samples. The discussion of these methods is performed on a set of in vivo confocal Raman spectra of human skin analysis that aims to identify skin aging markers. In the Appendix, a complete routine of data analysis is executed in a free software that can be used by the scientific community involved in these studies.

  16. Medial Cochlear Efferent Function: A Theoretical Analysis

    NASA Astrophysics Data System (ADS)

    Mountain, David C.

    2011-11-01

    Since the discovery of the cochlear efferent system, many hypotheses have been put forth for its function. These hypotheses for its function range from protecting the cochlea from over stimulation to improving the detection of sounds in noise. It is known that the medial efferent system innervates the outer hair cells and that stimulation of this system reduces basilar membrane and auditory nerve sensitivity which suggests that this system acts to decrease the gain of the cochlear amplifier. Here I present modeling results as well as analysis of published experimental data that suggest that the function of the medial efferent reflex is to decrease the cochlear amplifier gain by just the right amount so that the nonlinearity in the basilar membrane response lines up perfectly with the inner hair cell nonlinear transduction process to produce a hair cell receptor potential that is proportional to the logarithm of the sound pressure level.

  17. Industrial entrepreneurial network: Structural and functional analysis

    NASA Astrophysics Data System (ADS)

    Medvedeva, M. A.; Davletbaev, R. H.; Berg, D. B.; Nazarova, J. J.; Parusheva, S. S.

    2016-12-01

    Structure and functioning of two model industrial entrepreneurial networks are investigated in the present paper. One of these networks is forming when implementing an integrated project and consists of eight agents, which interact with each other and external environment. The other one is obtained from the municipal economy and is based on the set of the 12 real business entities. Analysis of the networks is carried out on the basis of the matrix of mutual payments aggregated over the certain time period. The matrix is created by the methods of experimental economics. Social Network Analysis (SNA) methods and instruments were used in the present research. The set of basic structural characteristics was investigated: set of quantitative parameters such as density, diameter, clustering coefficient, different kinds of centrality, and etc. They were compared with the random Bernoulli graphs of the corresponding size and density. Discovered variations of random and entrepreneurial networks structure are explained by the peculiarities of agents functioning in production network. Separately, were identified the closed exchange circuits (cyclically closed contours of graph) forming an autopoietic (self-replicating) network pattern. The purpose of the functional analysis was to identify the contribution of the autopoietic network pattern in its gross product. It was found that the magnitude of this contribution is more than 20%. Such value allows using of the complementary currency in order to stimulate economic activity of network agents.

  18. Automated volumetric stent analysis of in-vivo intracoronary optical coherence tomography three-dimensional datasets

    NASA Astrophysics Data System (ADS)

    Ughi, Giovanni J.; Adriaenssens, Tom; Onsea, Kevin; Dubois, Christophe; Coosemans, Mark; Sinnaeve, Peter; Desmet, Walter; D'hooge, Jan

    2011-06-01

    Intra-vascular Optical Coherence Tomography (IV-OCT) is an appropriate imaging modality for the evaluation of stent struts apposition and coverage in the coronary arteries. Most often, image analysis is performed by a time-consuming manual contour tracing process. Recently, we proposed an algorithm for fully automated lumen morphology and individual stent struts apposition/coverage quantification. In this manuscript further developments allowing for automatic segmentation of the stent contour are presented. As such, quantification of in-stent area, malapposition cross-sectional area (i.e. the area representing the space from the stent surface to the vessel wall) and coverage cross-sectional area (i.e. the area of the tissue covering the stent surface) are automatically obtained. Volumetric measurements of malapposition and coverage are then achieved through the analysis of equally-spaced consecutive IV-OCT cross-sectional images. In addition, uncovered and malapposed struts are automatically clustered through consecutive slices according to their three-dimensional spatial position. Finally, properties of each cluster (e.g. malapposition/coverage volumes and struts spatial location and distribution) are quantified allowing for a volumetric analysis of the implanted device. Validation of the algorithm was obtained taking as a reference manual measurements performed by an expert cardiologist. 102 in-vivo images, taken at random from 8 different patients, were both automatically and manually analyzed quantifying lumen and stent area. High Pearson's correlation coefficients (Rarea = 0.99) and Bland-Altman statistics, showing no significant bias and good limits of agreement, proved that the presented algorithm provides a robust and fast tool to automatically estimate apposition and coverage of stent through an entire in-vivo IV-OCT pullback. Such a tool will be important for the integration of this technology in clinical routine and large clinical trials.

  19. In vivo functional investigations of lactic acid in patients with respiratory chain disorders

    PubMed Central

    Touati, G; Rigal, O; Lombes, A; Frachon, P; Giraud, M; de Baulny, H O.

    1997-01-01

    Accepted 4 September 1996
 OBJECTIVE—To assess the prevalence of in vivo detectable abnormalities of lactate metabolism in mitochondrial disorders.
DESIGN—Retrospective study in a metabolic investigation unit.
PATIENTS—28 patients with a respiratory chain disorder identified from biochemical or genetic analyses, or both, and 133 age matched controls. Controls were children in whom causes of secondary hyperlactataemia and/or disorders, affecting the energy pathways could be excluded.
METHODS—Lactate and pyruvate were measured in blood, together with other intermediary metabolism indices, before and one hour after four meals each day. Lactate and creatinine in a 24 hour urine sample collected at the same time were analysed. When basal hyperlactataemia was not evident, an intravenous glucose or pyruvate loading test was performed as a provocative test.
RESULTS—Abnormal lactate metabolism was found in 25 of 28 patients thus demonstrating the potential usefulness of these investigations in the diagnosis of mitochondrial diseases. Moderate lactate accumulation was present in relatively mild disease, associated with a mitochondrial DNA mutation and combined respiratory complexes deficiency. By contrast, high lactate concentrations were observed in very young children, with severe disease, isolated complex deficiency, and no apparent mitochondrial DNA defect.

 PMID:9059154

  20. In vivo functional chronic imaging of a small animal model using optical-resolution photoacoustic microscopy

    PubMed Central

    Hu, Song; Maslov, Konstantin; Wang, Lihong V.

    2009-01-01

    Optical-resolution photoacoustic microscopy (OR-PAM) has been validated as a valuable tool for label-free volumetric microvascular imaging. More importantly, the advantages of noninvasiveness and measurement consistency suggest the use of OR-PAM for chronic imaging of intact microcirculation. Here, such chronic imaging is demonstrated for the first time by monitoring the healing process of laser-induced microvascular lesions in a small animal model in vivo. The central part of a 1 mm by 1 mm region in a nude mouse ear was treated under a continuous-wave laser to create a microvascular lesion for chronic study. The region of interest was imaged before the laser treatment, immediately after the treatment, and throughout the healing process using both the authors’ OR-PAM system and a commercial transmission-mode optical microscope. Three-dimensional microvascular morphology and blood oxygenation information were imaged simultaneously at capillary-level resolution. Transmission-mode optical microscopic images were acquired for comparison. OR-PAM has potential important applications in microcirculatory physiology or pathophysiology, tumor angiogenesis, laser microsurgery, and neuroscience. PMID:19610320

  1. A critical analysis of current in vitro and in vivo angiogenesis assays

    PubMed Central

    Staton, Carolyn A; Reed, Malcolm W R; Brown, Nicola J

    2009-01-01

    The study of angiogenesis has grown exponentially over the past 40 years with the recognition that angiogenesis is essential for numerous pathologies and, more recently, with the advent of successful drugs to inhibit angiogenesis in tumours. The main problem with angiogenesis research remains the choice of appropriate assays to evaluate the efficacy of potential new drugs and to identify potential targets within the angiogenic process. This selection is made more complex by the recognition that heterogeneity occurs, not only within the endothelial cells themselves, but also within the specific microenvironment to be studied. Thus, it is essential to choose the assay conditions and cell types that most closely resemble the angiogenic disease being studied. This is especially important when aiming to translate data from in vitro to in vivo and from preclinical to the clinic. Here we critically review and highlight recent advances in the principle assays in common use including those for endothelial cell proliferation, migration, differentiation and co-culture with fibroblasts and mural cells in vitro, vessel outgrowth from organ cultures and in vivo assays such as chick chorioallantoic membrane (CAM), zebrafish, sponge implantation, corneal, dorsal air sac, chamber and tumour angiogenesis models. Finally, we briefly discuss the direction likely to be taken in future studies, which include the use of increasingly sophisticated imaging analysis systems for data acquisition. PMID:19563606

  2. Comparison and Analysis of Toxcast Data with In Vivo Data for ...

    EPA Pesticide Factsheets

    The ToxCast program has generated a great wealth of in vitro high throughput screening (HTS) data on a large number of compounds, providing a unique resource of information on the bioactivity of these compounds. However, analysis of these data are ongoing, and interpretation and use of the ToxCast data such as for safety assessment of food related compounds remains undetermined. To fill this gap, we conducted a case study of 2 food-related compounds to better understand the ToxCast data and its potential use in chemical safety assessment by comparison between ToxCast and traditional, in vivo toxicology data using the Risk21 approach. Risk21 is an exposure driven flexible risk assessment framework developed by ILSI HESI. Prior work (Karmaus et. al., 2016) looking at all food-relevant compounds in ToxCast showed that food contact substances had high bioactivity in ToxCast assays. To better understand these chemicals based on their indirect food use, exposure and availability of traditional toxicology data, two compounds, dibutyltin dichloride and sodium pyrithione, were selected from a list of the food contact substances with the greatest activity in ToxCast. Exposure and hazard data were compiled and analyzed for both compounds. Comparison between in vitro HTS and in vivo data for sodium pyrithione showed that concentrations that elicited bioactivity in ToxCast assays corresponded to low- and no- observed adverse effect doses in animals. For dibutyltin dichlori

  3. In-vivo high resolution corneal imaging and analysis on animal models for clinical applications

    NASA Astrophysics Data System (ADS)

    Hong, Jesmond; Shinoj, V. K.; Murukeshan, V. M.; Baskaran, M.; Aung, Tin

    2015-07-01

    A simple and low cost optical probe system for the high resolution imaging of the cornea is proposed, based on a Gaussian beam epi-illumination configuration. Corneal topography is obtained by moving the scanning spot across the eye in a raster fashion whereas pachymetry data is achieved by reconstructing the images obtained at different depths. The proposed prototype has been successfully tested on porcine eye samples ex vivo and subsequently on laboratory animals, such as the New Zealand White Rabbit, in vivo. This proposed system and methodology pave the way for realizing a simple and inexpensive optical configuration for pachymetry and keratometry readings, with achievable resolution up to the cellular level. This novel and non-contact high resolution imaging modality demonstrates high intraobserver reproducibility and repeatability. Together with its sophisticated data analysis strategies and safety profile, it is believed to complement existing imaging modalities in the assessment and evaluation of corneal diseases, which enable a decrease in morbidity and improvement in the effectiveness of subsequent treatment.

  4. In vivo imaging of cell proliferation for a dynamic, whole body, analysis of undesired drug effects.

    PubMed

    Rizzi, Nicoletta; Manni, Isabella; Vantaggiato, Cristina; Delledonne, Giacomo Andrea; Gentileschi, Maria Pia; Maggi, Adriana; Piaggio, Giulia; Ciana, Paolo

    2015-06-01

    Noninvasive in vivo imaging offers a novel approach to preclinical studies opening the possibility of investigating biological events in the spatiotemporal dimension (eg, in any district of the body in time). Toxicological analysis may benefit from this novel approach through precise identification of the time and the target organs of toxicity manifestations, and assessment of the reversibility of toxic insults. The current limitation for routine application of this technology is the lack of appropriate surrogate markers for imaging toxicological events. Here, we demonstrate that in vivo imaging of a proliferation marker is capable of measuring the reduction of cell proliferation due to genotoxic/apoptotic agents, γ rays or antineoplastic drugs, or the increased proliferation associated with the inflammatory and regenerative reactions occurring after a toxic insult. A number of tools are currently available for imaging proliferation in preclinical and clinical settings, however our data provide a novel way to translate the evidence of toxic effects obtained in preclinical animal studies, by the direct, noninvasive measure of dividing cells in humans.

  5. Delayed near-infrared analysis permits visualization of rodent retinal pigment epithelium layer in vivo

    NASA Astrophysics Data System (ADS)

    Pankova, Natalie; Zhao, Xu; Liang, Huiyuan; Baek, David Sung Hyeon; Wang, Hai; Boyd, Shelley

    2014-07-01

    Patches of atrophy of the retinal pigment epithelium (RPE) have not been described in rodent models of retinal degeneration, as they have the clinical setting using fundus autofluorescence. We hypothesize that prelabeling the RPE would increase contrast and allow for improved visualization of RPE loss in vivo. Here, we demonstrate a new technique termed "delayed near-infrared analysis (DNIRA)" that permits ready detection of rat RPE, using optical imaging in the near-infrared (IR) spectrum with aid of indocyanine green (ICG) dye. Using DNIRA, we demonstrate a fluorescent RPE signal that is detected using confocal scanning laser ophthalmoscopy up to 28 days following ICG injection. This signal is apparent only after ICG injection, is dose dependent, requires the presence of the ICG filters (795/810 nm excitation/emission), does not appear in the IR reflectance channel, and is eliminated in the presence of sodium iodate, a toxin that causes RPE loss. Rat RPE explants confirm internalization of ICG dye. Together with normal retinal electrophysiology, these findings demonstrate that DNIRA is a new and safe noninvasive optical imaging technique for in vivo visualization of the RPE in models of retinal disease.

  6. Analysis of the in vivo confocal Raman spectral variability in human skin

    NASA Astrophysics Data System (ADS)

    Mogilevych, Borys; dos Santos, Laurita; Rangel, Joao L.; Grancianinov, Karen J. S.; Sousa, Mariane P.; Martin, Airton A.

    2015-06-01

    Biochemical composition of the skin changes in each layer and, therefore, the skin spectral profile vary with the depth. In this work, in vivo Confocal Raman spectroscopy studies were performed at different skin regions and depth profile (from the surface down to 10 μm) of the stratum corneum, to verify the variability and reproducibility of the intra- and interindividual Raman data. The Raman spectra were collected from seven healthy female study participants using a confocal Raman system from Rivers Diagnostic, with 785 nm excitation line and a CCD detector. Measurements were performed in the volar forearm region, at three different points at different depth, with the step of 2 μm. For each depth point, three spectra were acquired. Data analysis included the descriptive statistics (mean, standard deviation and residual) and Pearson's correlation coefficient calculation. Our results show that inter-individual variability is higher than intraindividual variability, and variability inside the SC is higher than on the skin surface. In all these cases we obtained r values, higher than 0.94, which correspond to high correlation between Raman spectra. It reinforces the possibility of the data reproducibility and direct comparison of in vivo results obtained with different study participants of the same age group and phototype.

  7. In vivo micro-CT analysis of bone remodeling in a rat calvarial defect model

    NASA Astrophysics Data System (ADS)

    Umoh, Joseph U.; Sampaio, Arthur V.; Welch, Ian; Pitelka, Vasek; Goldberg, Harvey A.; Underhill, T. Michael; Holdsworth, David W.

    2009-04-01

    The rodent calvarial defect model is commonly used to investigate bone regeneration and wound healing. This study presents a micro-computed tomography (micro-CT) methodology for measuring the bone mineral content (BMC) in a rat calvarial defect and validates it by estimating its precision error. Two defect models were implemented. A single 6 mm diameter defect was created in 20 rats, which were imaged in vivo for longitudinal experiments. Three 5 mm diameter defects were created in three additional rats, which were repeatedly imaged ex vivo to determine precision. Four control rats and four rats treated with bone morphogenetic protein were imaged at 3, 6, 9 and 12 weeks post-surgery. Scan parameters were 80 kVp, 0.45 mA and 180 mAs. Images were reconstructed with an isotropic resolution of 45 µm. At 6 weeks, the BMC in control animals (4.37 ± 0.66 mg) was significantly lower (p < 0.05) than that in treated rats (11.29 ± 1.01 mg). Linear regression between the BMC and bone fractional area, from 20 rats, showed a strong correlation (r2 = 0.70, p < 0.0001), indicating that the BMC can be used, in place of previous destructive analysis techniques, to characterize bone growth. The high precision (2.5%) of the micro-CT methodology indicates its utility in detecting small BMC changes in animals.

  8. Development of an In Vivo RNAi Protocol to Investigate Gene Function in the Filarial Nematode, Brugia malayi

    PubMed Central

    Song, Chuanzhe; Gallup, Jack M.; Day, Tim A.

    2010-01-01

    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. PMID:21203489

  9. Analysis of body calcium (regional changes in body calcium by in vivo neutron activation analysis)

    NASA Technical Reports Server (NTRS)

    Suki, W.; Johnson, P. C.; Leblanc, A.; Evans, H. J.

    1981-01-01

    The effect of space flight on urine and fecal calcium loss was documented during the three long-term Skylab flights. Neutron activation analysis was used to determine regional calcium loss. Various designs for regional analysis were investigated.

  10. Chemical analysis in vivo and in vitro by Raman spectroscopy – from single cells to humans

    PubMed Central

    Wachsmann-Hogiu, Sebastian; Weeks, Tyler

    2009-01-01

    Summary The gold standard for clinical diagnostics of tissues is immunofluorescence staining. Toxicity of many fluorescent dyes precludes their application in vivo. Raman spectroscopy, a chemically specific, label-free diagnostic technique, is rapidly gaining in acceptance as a powerful alternative. It has the ability to probe the chemical composition of biological materials in a nondestructive and mostly non-perturbing manner. We review the most recent developments in Raman spectroscopy in the life sciences, detailing advances in technology that have improved the ability to screen for diseases. Its role in the monitoring of biological function and mapping the intracellular chemical microenvironment will be discussed. Applications including endoscopy, surface-enhanced Raman scattering (SERS), and coherent Raman scattering (CRS) will be reviewed. PMID:19268566

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

    PubMed Central

    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

  12. Pharmacokinetic and toxicological evaluation of multi-functional thiol-6-fluoro-6-deoxy-d-glucose gold nanoparticles in vivo

    NASA Astrophysics Data System (ADS)

    Roa, Wilson; Xiong, Yeping; Chen, Jie; Yang, Xiaoyan; Song, Kun; Yang, Xiaohong; Kong, Beihua; Wilson, John; Xing, James Z.

    2012-09-01

    We synthesized a novel, multi-functional, radiosensitizing agent by covalently linking 6-fluoro-6-deoxy-d-glucose (6-FDG) to gold nanoparticles (6-FDG-GNPs) via a thiol functional group. We then assessed the bio-distribution and pharmacokinetic properties of 6-FDG-GNPs in vivo using a murine model. At 2 h, following intravenous injection of 6-FDG-GNPs into the murine model, approximately 30% of the 6-FDG-GNPs were distributed to three major organs: the liver, the spleen and the kidney. PEGylation of the 6-FDG-GNPs was found to significantly improve the bio-distribution of 6-FDG-GNPs by avoiding unintentional uptake into these organs, while simultaneously doubling the cellular uptake of GNPs in implanted breast MCF-7 adenocarcinoma. When combined with radiation, PEG-6-FDG-GNPs were found to increase the apoptosis of the MCF-7 breast adenocarinoma cells by radiation both in vitro and in vivo. Pharmacokinetic data indicate that GNPs reach their maximal concentrations at a time window of two to four hours post-injection, during which optimal radiation efficiency can be achieved. PEG-6-FDG-GNPs are thus novel nanoparticles that preferentially accumulate in targeted cancer cells where they act as potent radiosensitizing agents. Future research will aim to substitute the 18F atom into the 6-FDG molecule so that the PEG-6-FDG-GNPs can also function as radiotracers for use in positron emission tomography scanning to aid cancer diagnosis and image guided radiation therapy planning.

  13. MOOD STATES, SYMPATHETIC ACTIVITY, AND IN VIVO β-ADRENERGIC RECEPTOR FUNCTION IN A NORMAL POPULATION

    PubMed Central

    Yu, Bum-Hee; Kang, Eun-Ho; Ziegler, Michael G.; Mills, Paul J.; Dimsdale, Joel E.

    2009-01-01

    The purpose of this study was to examine the relationship between mood states and β-adrenergic receptor function in a normal population. We also examined if sympathetic nervous system activity is related to mood states or β-adrenergic receptor function. Sixty-two participants aged 25–50 years were enrolled in this study. Mood states were assessed using the Profile of Mood States (POMS). β-adrenergic receptor function was determined using the chronotropic 25 dose isoproterenol infusion test. Level of sympathetic nervous system activity was estimated from 24-hr urine norepinephrine excretion. Higher tension-anxiety, depression-dejection, and anger-hostility were related to decreased β-adrenergic receptor sensitivity (i.e., higher chronotropic 25 dose values), but tension-anxiety was the only remaining independent predictor of β-adrenergic receptor function after controlling for age, gender, ethnicity, and body mass index (BMI). Urinary norepinephrine excretion was unrelated to either mood states or β-adrenergic receptor function. These findings replicate previous reports that anxiety is related to decreased (i.e., desensitized) β-adrenergic receptor sensitivity, even after controlling for age, gender, ethnicity, and body mass index. PMID:17583588

  14. Chlamydia trachomatis ChxR is a transcriptional regulator of virulence factors that function in in vivo host pathogen interactions.

    PubMed

    Yang, Chunfu; Kari, Laszlo; Sturdevant, Gail L; Song, Lihua; Patton, Michael John; Couch, Claire E; Ilgenfritz, Jillian M; Southern, Timothy R; Whitmire, William M; Briones, Michael; Bonner, Christine; Grant, Chris; Hu, Pinzhao; McClarty, Grant; Caldwell, Harlan D

    2017-03-22

    Chlamydia trachomatis is an obligate intracellular pathogen characterized by a unique biphasic developmental cycle that alternates between infectious and non-infectious organisms. Chlamydial ChxR is a transcriptional activator that has been implicated in the regulation of the development cycle. We used a reverse genetics approach to generate three chxR null mutants. All three mutants grew normally in cultured mammalian cells. Whole genome sequencing identified SNPs in other genes, however, none of the mutated genes were common to all three ChxR null mutants arguing against a genetic compensatory mechanism that would explain the non-essential in vitro growth phenotype. Comparative proteomics identified five proteins, CT005, CT214, CT565, CT694 and CT695 that were significantly down regulated in all ChxR null mutants. This group includes established inclusion membrane and type III secreted proteins. ChxR transcriptional regulation of these genes was confirmed by qRT-PCR. Importantly, while ChxR null mutants exhibited no growth deficiencies in in vitro, they did show significant differences in in vivo growth using a mouse genital tract model. Collectively, our findings demonstrated that ChxR is a transcriptional activator that regulates the expression of virulence genes whose functions are restricted to in vivo infection.

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

    SciTech Connect

    Teplova, Marianna; Hafner, Markus; Teplov, Dmitri; Essig, Katharina; Tuschl, Thomas; Patel, Dinshaw J.

    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.

  16. Optimization of a Model Corrected Blood Input Function from Dynamic FDG-PET Images of Small Animal Heart In Vivo

    PubMed Central

    Zhong, Min; Kundu, Bijoy K.

    2013-01-01

    Quantitative evaluation of dynamic Positron Emission Tomography (PET) of mouse heart in vivo is challenging due to the small size of the heart and limited intrinsic spatial resolution of the PET scanner. Here, we optimized a compartment model which can simultaneously correct for spill over and partial volume effects for both blood pool and the myocardium, compute kinetic rate parameters and generate model corrected blood input function (MCBIF) from ordered subset expectation maximization – maximum a posteriori (OSEM-MAP) cardiac and respiratory gated 18F-FDG PET images of mouse heart with attenuation correction in vivo, without any invasive blood sampling. Arterial blood samples were collected from a single mouse to indicate the feasibility of the proposed method. In order to establish statistical significance, venous blood samples from n=6 mice were obtained at 2 late time points, when SP contamination from the tissue to the blood is maximum. We observed that correct bounds and initial guesses for the PV and SP coefficients accurately model the wash-in and wash-out dynamics of the tracer from mouse blood. The residual plot indicated an average difference of about 1.7% between the blood samples and MCBIF. The downstream rate of myocardial FDG influx constant, Ki (0.15±0.03 min−1), compared well with Ki obtained from arterial blood samples (P=0.716). In conclusion, the proposed methodology is not only quantitative but also reproducible. PMID:24741130

  17. Extensive Ex Vivo Expansion of Functional Human Erythroid Precursors Established From Umbilical Cord Blood Cells by Defined Factors

    PubMed Central

    Huang, Xiaosong; Shah, Siddharth; Wang, Jing; Ye, Zhaohui; Dowey, Sarah N; Tsang, Kit Man; Mendelsohn, Laurel G; Kato, Gregory J; Kickler, Thomas S; Cheng, Linzhao

    2014-01-01

    There is a constant shortage of red blood cells (RBCs) from sufficiently matched donors for patients who need chronic transfusion. Ex vivo expansion and maturation of human erythroid precursors (erythroblasts) from the patients or optimally matched donors could represent a potential solution. Proliferating erythroblasts can be expanded from umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 106–107-fold (in ~50 days) before proliferation arrest and reaching sufficient number for broad application. Here, we report that ectopic expression of three genetic factors (Sox2, c-Myc, and an shRNA against TP53 gene) associated with iPSC derivation enables CB-derived erythroblasts to undergo extended expansion (~1068-fold in ~12 months) in a serum-free culture condition without change of cell identity or function. These expanding erythroblasts maintain immature erythroblast phenotypes and morphology, a normal diploid karyotype and dependence on a specific combination of growth factors for proliferation throughout expansion period. When being switched to a terminal differentiation condition, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes that can bind and release oxygen. Our result may ultimately lead to an alternative approach to generate unlimited numbers of RBCs for personalized transfusion medicine. PMID:24002691

  18. In Vitro and In Vivo Evaluation of Tetracycline Loaded Chitosan-Gelatin Nanosphere Coatings for Titanium Surface Functionalization.

    PubMed

    Ma, Kena; Cai, Xinjie; Zhou, Yi; Wang, Yining; Jiang, Tao

    2017-02-01

    Owing to the biocompatibility of titanium surface, titanium implants are suitable substrates for microbial colonization and biofilm formation, which is still a serious clinical threat. Current research trends have been focused on the development of antibacterial coatings on titanium substrate or adhesion resistant surface. In our previous study, tetracycline (Tc) loaded chitosan-gelatin (CSG) nanosphere coatings are successfully fabricated on titanium substrates via electrophoretic deposition. These coatings show nanosphere structure, and excellent antibacterial property in vitro. However, further in vitro and in vivo evaluation of the coatings is required for the future application. Therefore, in the present study, the authors investigate the coatings' mechanical, swelling and degradation property, in vitro cellular response to preosteoblast cells, and the antibacterial property in rabbits. Results show that Tc incorporation can improve the tensile bond strength of the coating, decrease the swelling ratio, and accelerate the degradation of the coating. Although high Tc concentration group exhibits cytotoxicity to MC3T3-E1 cells, its in vivo antibacterial property is preferred, and shows better outcome than the prophylactic administration of Tc. Tc loaded CSG nanosphere coatings are suitable antibacterial coatings for titanium surface functionalization.

  19. [Suppressive effects of lanoconazole on arthus phenomenon in vivo and on production and functions of TNF in vitro].

    PubMed

    Mitsuya, M; Wada, K; Ishibashi, H; Tansho, S; Abe, S; Yamaguchi, H

    2000-01-01

    The anti-inflammatory effect of lanoconazole (LCZ) was investigated in vivo and in vitro. The effect of LCZ was evaluated on the inflammatory reactions elicited by intradermal injection of ovalbumin to ovalbumin-immunized rabbits, as an Arthus phenomenon. A one or two % cream preparation of LCZ was topically applied on the lesion daily after challenging injection until the inflamation had diminished. By macroscopic observation and measuring the diameter of edema, erythema, hemorrhage and necrosis, the effects of LCZ on the reactions were compared with the reactions of the sites administered withcream vehicle as reference agent. Two % LCZ showed an anti-hemorrhagic effect. The in vitro effect of LCZ on production and functions of an inflammatory cytokine, TNF was also examined. LCZ suppressed the production of TNF by murine peritoneal macrophages at 20 micro g/ml and the adhesion of neutrophils at 100 micro g/ml. Moreover, LCZ significantly suppressed the growth inhibitory activity of TNF against L929 fibroblasts at 0.5 micro g/ml. A very low concentration of LCZ might protect the fibroblasts from immunological cytotoxicity in vivo. These findings suggest that LCZ has a suppressive activity to inflammatory responses and this suppressive action may be due to its protective activity to cells like fibroblasts.

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

    PubMed Central

    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

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

    PubMed

    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

  2. In vivo mitochondrial inhibition alters corticostriatal synaptic function and the modulatory effects of neurotrophins.

    PubMed

    Mendoza, E; Miranda-Barrientos, J A; Vázquez-Roque, R A; Morales-Herrera, E; Ruelas, A; De la Rosa, G; Flores, G; Hernández-Echeagaray, E

    2014-11-07

    Experimental evidence has revealed the role of mitochondria in various aspects of neuronal physiology. Mitochondrial failure results in alterations that underlie the pathogeneses of many neurodegenerative disorders, such as Parkinson's disease, Alzheimer's disease, Huntington's disease (HD) and amyotrophic lateral sclerosis. The mitochondrial toxin 3-nitropropionic acid (3-NP) has been used to model failure; for example, systemic administration of 3-NP imitates the striatal degeneration that is exhibited in the postmortem tissue of patients afflicted with HD. We have demonstrated that low, sub-chronic doses of 3-NP are sufficient to initiate the damage to striatal neurons that is associated with changes in neurotrophin expression levels. However, the mechanisms underlying the alterations in neuronal activity and neurotransmission due to 3-NP-induced mitochondrial dysfunction remain to be elucidated. In this paper, we focus on how corticostriatal transmission and its modulation by neurotrophins are altered in vivo after 5 days of mitochondrial inhibition with 3-NP. Recordings of population spikes and a paired pulse (PP) stimulation protocol were used to document changes in corticostriatal synapses in 3-NP-treated brain slices. The corticostriatal synapses were modulated by neurotrophins but displayed differential amplitude increases in the presence of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4/5 (NT-4/5) under control conditions. Neurotrophin-mediated synaptic modulation was decreased in slices from 3-NP-treated mice. The protein and mRNA levels of neurotrophins and their receptors were also modified in the 3-NP-treated tissue. Neuronal structural evaluation demonstrated that synaptic length and density were reduced in the 3-NP-treated mice, which partially explained the changes in the amplitudes of the synaptic field responses. Our results demonstrate that corticostriatal synapses are differentially modulated by neurotrophins

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

    PubMed

    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.

  4. BEN domain protein Elba2 can functionally substitute for linker histone H1 in Drosophila in vivo

    PubMed Central

    Xu, Na; Lu, Xingwu; Kavi, Harsh; Emelyanov, Alexander V.; Bernardo, Travis J.; Vershilova, Elena; Skoultchi, Arthur I.; Fyodorov, Dmitry V.

    2016-01-01

    Metazoan linker histones are essential for development and play crucial roles in organization of chromatin, modification of epigenetic states and regulation of genetic activity. Vertebrates express multiple linker histone H1 isoforms, which may function redundantly. In contrast, H1 isoforms are not present in Dipterans, including D. melanogaster, except for an embryo-specific, distantly related dBigH1. Here we show that Drosophila BEN domain protein Elba2, which is expressed in early embryos and was hypothesized to have insulator-specific functions, can compensate for the loss of H1 in vivo. Although the Elba2 gene is not essential, its mutation causes a disruption of normal internucleosomal spacing of chromatin and reduced nuclear compaction in syncytial embryos. Elba2 protein is distributed ubiquitously in polytene chromosomes and strongly colocalizes with H1. In H1-depleted animals, ectopic expression of Elba2 rescues the increased lethality and ameliorates abnormalities of chromosome architecture and heterochromatin functions. We also demonstrate that ectopic expression of BigH1 similarly complements the deficiency of H1 protein. Thus, in organisms that do not express redundant H1 isoforms, the structural and biological functions performed by canonical linker histones in later development, may be shared in early embryos by weakly homologous proteins, such as BigH1, or even unrelated, non-homologous proteins, such as Elba2. PMID:27687115

  5. Measurements of fluorine in contemporary urban Canadians: a comparison of the levels found in human bone using in vivo and ex vivo neutron activation analysis.

    PubMed

    Mostafaei, F; McNeill, F E; Chettle, D R; Wainman, B C; Pidruczny, A E; Prestwich, W V

    2015-03-01

    Non-invasive in vivo neutron activation analysis (NAA) was used to measure the fluorine concentration in 35 people in Hamilton, Ontario, Canada. Measurement and precision data of this second generation NAA system were determined in 2013, and the results were compared with the performance of a first generation system used in a pilot study of 33 participants from the Hamilton area in 2008. Improvements in precision in line with those predicted by phantom studies were observed, but the use of fewer technicians during measurement seemed adversely to affect performance. We compared the levels of fluorine observed in people between the two studies and found them to be comparable. The average fluorine concentration in bone was found to be 3  ±  0.3 mg and 3.5  ±  0.4 mg F/g Ca for 2013 and 2008 measurements respectively. Ten people were measured in both studies; the observed average change in bone fluorine in this subgroup was consistent with that predicted by the observation of the relationship between bone fluorine and age in the wider group. In addition, we observed differences in the relationship between bone fluorine level and age between men and women, which may be attributable either to sex or gender differences. The rate of increase in fluorine content for men was found to be 0.096  ±  0.022 mg F/g Ca per year while the rate of increase for women was found to be slightly less than half that of men, 0.041  ±  0.017 mg F/g Ca per year. A discontinuity in the rate of increase in fluorine content with age was observed in women at around age 50. Bone fluorine content was significantly lower ([Formula: see text]) in women age 50 to 59 than in women age 40 to 49, which we suggest may be attributable to bone metabolism changes associated with menopause. We also observed increased fluorine levels in tea drinkers as compared to non-tea drinkers, suggesting tea may be a significant source of exposure in Canada. The rate of increase in fluorine

  6. Proliferation of Functional Hair Cells in Vivo in the Absence of the Retinoblastoma Protein

    NASA Astrophysics Data System (ADS)

    Sage, Cyrille; Huang, Mingqian; Karimi, Kambiz; Gutierrez, Gabriel; Vollrath, Melissa A.; Zhang, Duan-Sun; García-Añoveros, Jaime; Hinds, Philip W.; Corwin, Jeffrey T.; Corey, David P.; Chen, Zheng-Yi

    2005-02-01

    In mammals, hair cell loss causes irreversible hearing and balance impairment because hair cells are terminally differentiated and do not regenerate spontaneously. By profiling gene expression in developing mouse vestibular organs, we identified the retinoblastoma protein (pRb) as a candidate regulator of cell cycle exit in hair cells. Differentiated and functional mouse hair cells with a targeted deletion of Rb1 undergo mitosis, divide, and cycle, yet continue to become highly differentiated and functional. Moreover, acute loss of Rb1 in postnatal hair cells caused cell cycle reentry. Manipulation of the pRb pathway may ultimately lead to mammalian hair cell regeneration.

  7. Keratoglobus and posterior subcapsular cataract: surgical considerations and in vivo microstructural analysis.

    PubMed

    Ku, Judy Y F; Grupcheva, Christina N; Fisk, Michael J; McGhee, Charles N J

    2004-01-01

    We report sporadic, bilateral keratoglobus associated with posterior subcapsular cataract in a 43-year-old man. Slitlamp biomicroscopy showed symmetric arcus senilis-like deposits, a polygonal appearance resembling crocodile shagreen, an unusual endothelial appearance, and posterior subcapsular cataract. Orbscan II pachymetry maps (Bausch & Lomb) demonstrated bilateral diffuse corneal thinning (359.53 microm +/- 21.15 [SD] in the right eye and 379.61 +/- 11.49 microm in the left eye). These thickness values were confirmed by ultrasound pachymetry. In vivo confocal microscopy showed multiple criss-crossing dark lines and no identifiable cellular elements within the stroma. There were mild to moderate, guttata-like endothelial changes surrounded by pleomorphic cells. Phacoemulsification was performed in the left eye after careful consideration of the presenting features and modification of the surgical technique. Minimal structural alteration was observed during microstructural analysis 7 months after surgery. The endothelial morphology postoperatively was similar to that at baseline.

  8. Optical barcoding of PLGA for multispectral analysis of nanoparticle fate in vivo.

    PubMed

    Medina, David X; Householder, Kyle T; Ceton, Ricki; Kovalik, Tina; Heffernan, John M; Shankar, Rohini V; Bowser, Robert P; Wechsler-Reya, Robert J; Sirianni, Rachael W

    2017-03-03

    Understanding of the mechanisms by which systemically administered nanoparticles achieve delivery across biological barriers remains incomplete, due in part to the challenge of tracking nanoparticle fate in the body. Here, we develop a new approach for "barcoding" nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) with bright, spectrally defined quantum dots (QDs) to enable direct, fluorescent detection of nanoparticle fate with subcellular resolution. We show that QD labeling does not affect major biophysical properties of nanoparticles or their interaction with cells and tissues. Live cell imaging enabled simultaneous visualization of the interaction of control and targeted nanoparticles with bEnd.3 cells in a flow chamber, providing direct evidence that surface modification of nanoparticles with the cell-penetrating peptide TAT increases their biophysical association with cell surfaces over very short time periods under convective current. We next developed this technique for quantitative biodistribution analysis in vivo. These studies demonstrate that nanoparticle surface modification with the cell penetrating peptide TAT facilitates brain-specific delivery that is restricted to brain vasculature. Although nanoparticle entry into the healthy brain parenchyma is minimal, with no evidence for movement of nanoparticles across the blood-brain barrier (BBB), we observed that nanoparticles are able to enter to the central nervous system (CNS) through regions of altered BBB permeability - for example, into circumventricular organs in the brain or leaky vasculature of late-stage intracranial tumors. In sum, these data demonstrate a new, multispectral approach for barcoding PLGA, which enables simultaneous, quantitative analysis of the fate of multiple nanoparticle formulations in vivo.

  9. Functional Analysis of Arabidopsis Sucrose Transporters

    SciTech Connect

    John M. Ward

    2009-03-31

    Sucrose is the main photosynthetic product that is transported in the vasculature of plants. The long-distance transport of carbohydrates is required to support the growth and development of net-importing (sink) tissues such as fruit, seeds and roots. This project is focused on understanding the transport mechanism sucrose transporters (SUTs). These are proton-coupled sucrose uptake transporters (membrane proteins) that are required for transport of sucrose in the vasculature and uptake into sink tissues. The accomplishments of this project included: 1) the first analysis of substrate specificity for any SUT. This was accomplished using electrophysiology to analyze AtSUC2, a sucrose transporter from companion cells in Arabidopsis. 2) the first analysis of the transport activity for a monocot SUT. The transport kinetics and substrate specificity of HvSUT1 from barley were studied. 3) the first analysis of a sucrose transporter from sugarcane. and 4) the first analysis of transport activity of a sugar alcohol transporter homolog from plants, AtPLT5. During this period four primary research papers, funded directly by the project, were published in refereed journals. The characterization of several sucrose transporters was essential for the current effort in the analysis of structure/function for this gene family. In particular, the demonstration of strong differences in substrate specificity between type I and II SUTs was important to identify targets for site-directed mutagenesis.

  10. Applying microscopy to the analysis of nuclear structure and function.

    PubMed

    Iborra, Francisco; Cook, Peter R; Jackson, Dean A

    2003-02-01

    One of the ultimate goals of biological research is to understand mechanisms of cell function within living organisms. With this in mind, many sophisticated technologies that allow us to inspect macromolecular structure in exquisite detail have been developed. Although knowledge of structure derived from techniques such as X-ray crystallography and nuclear magnetic resonance is of vital importance, these approaches cannot reveal the remarkable complexity of molecular interactions that exists in vivo. With this in mind, this review focuses on the use of microscopy techniques to analyze cell structure and function. We describe the different basic microscopic methodologies and how the routine techniques are best applied to particular biological problems. We also emphasize the specific capabilities and uses of light and electron microscopy and highlight their individual advantages and disadvantages. For completion, we also comment on the alternative possibilities provided by a variety of advanced imaging technologies. We hope that this brief analysis of the undoubted power of microscopy techniques will be enough to stimulate a wider participation in this rapidly developing area of biological discovery.

  11. Docosahexaenoic acid and phosphatidylserine improves the antioxidant activities in vitro and in vivo and cognitive functions of the developing brain.

    PubMed

    Chaung, Hso-Chi; Chang, Chin-Dong; Chen, Pi-Hang; Chang, Chia-Jung; Liu, Shyh-Hwa; Chen, Chih-Cheng

    2013-05-01

    Fish oil during early postnatal period may modulate the impact of oxidative stress in the developing brain and thus improve memory and cognitive behaviour. This study investigated the impacts of docosahexaenoic acid (DHA, C22:6, n-3) and/or phosphatidylserine (PS) on antioxidant activities in vitro, and the beneficial effects of feeding with DHA and/or PS on antioxidant activities in brain and liver tissues and on the cognitive functions of the developing brain. Results indicated that DHA and/or PS significantly enhanced antioxidant activities and increased cell viabilities in vitro. Feeding with DHA and/or PS supplementation not only significantly improved escape latency of animals, but it also improved the oxidative parameters in the brain, enhanced glutathione peroxidase activity as well as reduced nitric mono-oxide levels in the liver. DHA and PS may serve to protect cells from oxidative stress and further improve learning and memory ability in vivo.

  12. Nodular lymphoid hyperplasia of the bowel in primary hypogammaglobulinaemia: study of in vivo and in vitro lymphocyte function.

    PubMed Central

    Webster, A D; Kenwright, S; Ballard, J; Shiner, M; Slavin, G; Levi, A J; Loewi, G; Asherson, G L

    1977-01-01

    In vitro and in vivo lymphocyte function was studied in six patients with primary hypogammaglobulinaemia and nodular lymphoid hyperplasia (NLH) of the bowel. Lymphocyte transformation, numbers of circulating T and B lymphocytes, and delayed hypersensitivity skin tests did not significantly differ when compared with hypogammaglobulinaemic patients without NLH. However, patients with NLH had higher jejunal juice IgM concentrations and a tendency to higher serum IgM concentrations than those without NLH. The morphological features of NLH are similar to the germinal centres of lymph nodes but more closely resemble the follicle zone of Peyer's patches. These findings suggest that NLH represents a local immune response to antigens originating in the gut lumen. Images Fig. 1 Fig. 2 Fig. 3(a)-3(b) Fig. 3(c) Fig. 4 Fig. 5 Fig. 6 PMID:873321

  13. Lack of Functionally-Active Sweet Taste Receptors in the Jejunum in vivo in the Rat

    PubMed Central

    Chaudhry, Rizwan M.; Garg, Alok; Abdelfatah, Mohamed M.; Duenes, Judith A.; Sarr, Michael G.

    2013-01-01

    BACKGROUND When studied in enterocyte-like cell lines (Caco-2 and RIE cells), agonists and antagonists of the sweet taste receptor (STR) augment and decrease glucose uptake, respectively. We hypothesize that exposure to STR agonists and antagonists in vivo will augment glucose absorption in the rat. MATERIAL/METHODS 30-cm segments of jejunum in anesthetized rats were perfused with iso-osmolar solutions containing 10, 35, and 100 mM glucose solutions (n=6 rats, each group) with and without the STR agonist 2 mM acesulfame potassium (AceK) and the STR inhibitor 10 μM U-73122 (inhibitor of the PLC pathway). Carrier-mediated absorption of glucose was calculated by using stereospecific and non-stereospecific 14C-D-glucose and 3H-L-glucose, respectively. RESULTS Addition of the STR agonist AceK to the 10, 35, and 100 mM glucose solutions had no substantive effects on glucose absorption from 2.1±0.2 to 2.0±0.3, 5.8±0.2 to 4.8±0.2, and 15.5±2.3 to 15.7±2.7 μmol/min/30-cm intestinal segment (p>0.05), respectively. Addition of the STR inhibitor (U-73122) also had no effect on absorption in the 10, 35, and 100 mM solutions from 2.3±0.1 to 2.1±0.2, 7.7±0.5 to 7.2±0.5, and 15.7±0.9 to 15.2±1.1 μmol/min/30-cm intestinal segment, respectively. CONCLUSION Provision of glucose directly into rat jejunum does not augment glucose absorption via STR-mediated mechanisms within the jejunum in the rat. Our experiments show either no major role of STRs in mediating postprandial augmentation of glucose absorption or that proximal gastrointestinal tract stimulation of STR or other luminal factors may be required for absorption of glucose to be augmented by STR. PMID:23531453

  14. Soy protein concentrate mitigates markers of colonic inflammation and loss of gut barrier function in vitro and in vivo.

    PubMed

    Bitzer, Zachary T; Wopperer, Amy L; Chrisfield, Benjamin J; Tao, Ling; Cooper, Timothy K; Vanamala, Jairam; Elias, Ryan J; Hayes, John E; Lambert, Joshua D

    2017-02-01

    Whereas a number of studies have examined the effects of soy isoflavones and tocopherols on colonic inflammation, few have examined soy protein. We determined the radical scavenging and cytoprotective effects of soy protein concentrate (SPC) in vitro and its anti-inflammatory effects in dextran sulfate sodium (DSS)-treated mice. Cotreatment with SPC protected Caco-2 human colon cells from H2O2-induced cell death and mitigated intracellular oxidative stress. Treatment of differentiated Caco-2 cells with SPC blunted DSS-induced increases in monolayer permeability. Pepsin/pancreatin-digested SPC had reduced radical scavenging activity, but retained the monolayer protective effects of SPC. In vivo, 1.5% DSS caused body weight loss, colon shortening, and splenomegaly in CF-1 mice. Co-treatment with 12% SPC mitigated DSS-induced body weight loss and splenomegaly. DSS increased colonic interleukin (IL)-1β, IL-6, and monocyte chemotactic protein-1 expression. The levels of these markers were significantly lower in mice co-treated with SPC. SPC prevented DSS-mediated reductions in colonic glucagon-like peptide 2 levels, suggesting that SPC can prevent loss of gut barrier function, but no significant effect on claudin 1 and occludin mRNA levels of was observed. SPC-treated mice had lower colonic mRNA expression of toll-like receptor 4 and nucleotide-binding oligomerization domain-containing protein-like receptor family, pyrin domain containing protein 3 (NLRP3), and lower caspase-1 enzyme activity than DSS-treated mice. In summary, SPC exerted antioxidant and cytoprotective effects in vitro and moderated the severity of DSS-induced inflammation and loss of gut barrier function in vivo. These effects appear to be mediated in part through reduced NLRP3 expression and caspase 1 activity.

  15. Enhanced Control of In Vivo Bone Formation with Surface Functionalized Alginate Microbeads Incorporating Heparin and Human Bone Morphogenetic Protein-2

    PubMed Central

    Abbah, Sunny Akogwu; Liu, Jing; Goh, James Cho Hong

    2013-01-01

    In this study, we tested the hypothesis that a surface functionalization delivery platform incorporating heparin onto strontium alginate microbeads surfaces would convert this “naive carriers” into “mini-reservoirs” for localized in vivo delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2) that will induce functional bone regeneration. In vitro evaluation confirmed that (1) heparin incorporation could immobilize and prolong rhBMP-2 release for approximately 3 weeks; (2) a significant decrease (p<0.01) in rhBMP-2 burst release is attainable depending on initial protein load; and (3) rhBMP-2 released from surface functionalized microbeads retained bioactivity and stimulated higher alkaline phosphatase activity in cultured C2C12 cells when compared with daily administration of fresh bolus rhBMP-2. Subsequently, surface functionalized microbeads were used for in vivo delivery of rhBMP-2 at local sites of posterolateral spinal fusion surgery in rats. The microbeads were loaded into the pores of medical-grade polyepsilone caprolactone-tricalcium phosphate scaffolds before implantation. Results revealed robust bone formation and a biomechanically solid fusion after 6 weeks. When compared with a control group consisting of an equivalent amount of rhBMP-2 that was directly adsorbed onto bare-surfaced microbeads with no heparin, a 5.3-fold increase in bone volume fraction and a 2.6-fold increase in bending stiffness (flexion/extension) were observed. When compared with collagen sponge carriers of rhBMP-2, a 1.5-fold and a 1.3-fold increase in bone volume fraction and bending stiffness were observed, respectively. More importantly, 3D micro-computed tomography images enabled the visualization of a well-contained newly formed bone at ipsilateral implant sites with surface functionalized rhBMP-2 delivery. This was absent with collagen sponge carriers where newly formed bone tissue was poorly contained and crossed over the posterior midline to

  16. Four-dimensional analysis of stomatognathic function.

    PubMed

    Terajima, Masahiko; Endo, Mizuki; Aoki, Yoshimitsu; Yuuda, Kyouko; Hayasaki, Haruaki; Goto, Tazuko K; Tokumori, Kenji; Nakasima, Akihiko

    2008-08-01

    Many researchers have attempted to clarify the complex relationships between stomatognathic function and craniofacial morphology. Most studies investigated the trajectories of incisal or condylar points and measured temporomandibular morphology projected onto 2-dimensional radiographic films. Although these methods provided valuable information, their diagnostic capabilities were limited. We introduce a new 4-dimensional (4D) analysis of stomatognathic function that combines the 3-dimensional (3D) computed tomography of the cranium and mandible, dental surface imaging with a noncontact 3D laser scanner, and mandibular movement data recorded with a 6 degrees of freedom jaw-movement analyzer. This method performs dynamic and precise simulations that can analyze and display condyle to fossa distances and occlusal contacts during mandibular function. These comprehensive relationships can be analyzed and displayed not only at intercuspal position, but also at any mandibular position during functional movements. We believe that our 4D analyzing system will be useful for diagnosing temporomandibular disorders of patients with jaw deformities and other malocclusions.

  17. In vivo maturation of functional renal organoids formed from embryonic cell suspensions.

    PubMed

    Xinaris, Christodoulos; Benedetti, Valentina; Rizzo, Paola; Abbate, Mauro; Corna, Daniela; Azzollini, Nadia; Conti, Sara; Unbekandt, Mathieu; Davies, Jamie A; Morigi, Marina; Benigni, Ariela; Remuzzi, Giuseppe

    2012-11-01

    The shortage of transplantable organs provides an impetus to develop tissue-engineered alternatives. Producing tissues similar to immature kidneys from simple suspensions of fully dissociated embryonic renal cells is possible in vitro, but glomeruli do not form in the avascular environment. Here, we constructed renal organoids from single-cell suspensions derived from E11.5 kidneys and then implanted these organoids below the kidney capsule of a living rat host. This implantation resulted in further maturation of kidney tissue, formation of vascularized glomeruli with fully differentiated capillary walls, including the slit diaphragm, and appearance of erythropoietin-producing cells. The implanted tissue exhibited physiologic functions, including tubular reabsorption of macromolecules, that gained access to the tubular lumen on glomerular filtration. The ability to generate vascularized nephrons from single-cell suspensions marks a significant step to the long-term goal of replacing renal function by a tissue-engineered kidney.

  18. In Vivo Maturation of Functional Renal Organoids Formed from Embryonic Cell Suspensions

    PubMed Central

    Benedetti, Valentina; Rizzo, Paola; Abbate, Mauro; Corna, Daniela; Azzollini, Nadia; Conti, Sara; Unbekandt, Mathieu; Davies, Jamie A.; Morigi, Marina; Benigni, Ariela; Remuzzi, Giuseppe

    2012-01-01

    The shortage of transplantable organs provides an impetus to develop tissue-engineered alternatives. Producing tissues similar to immature kidneys from simple suspensions of fully dissociated embryonic renal cells is possible in vitro, but glomeruli do not form in the avascular environment. Here, we constructed renal organoids from single-cell suspensions derived from E11.5 kidneys and then implanted these organoids below the kidney capsule of a living rat host. This implantation resulted in further maturation of kidney tissue, formation of vascularized glomeruli with fully differentiated capillary walls, including the slit diaphragm, and appearance of erythropoietin-producing cells. The implanted tissue exhibited physiologic functions, including tubular reabsorption of macromolecules, that gained access to the tubular lumen on glomerular filtration. The ability to generate vascularized nephrons from single-cell suspensions marks a significant step to the long-term goal of replacing renal function by a tissue-engineered kidney. PMID:23085631

  19. The Function of CTLA4 During the In Vivo Immune Response to Infectious Disease

    DTIC Science & Technology

    2000-09-20

    CONTENTS I. Introduction A. The two-signal model ofT cell activation B. CTLA4 and CD28 structure and expression C. CTLA4 function D. CTLA4 mechanism ...infectious disease models. and to address the molecular mechanism ofCTLA4 signaling. In this chapter. I \\\\’ill discuss CTLA4 structure and expression. and...and some conflicting data that remain to be resolved. Current knowledge of the mechanism of CTLA4 inhibitory action and CTLA4-mediated signal

  20. Effects of systemic glucocorticosteroids on peripheral neutrophil functions in asthmatic subjects: an ex vivo study

    PubMed Central

    Bancalari, L.; Giannessi, D.; Bernini, W.; Lazzerini, G.; Sicari, R.; Bacci, E.; Dente, F. L.; Vagaggini, B.; Caterina, R. De

    1995-01-01

    In 21 asthmatic subjects, several functions of isolated peripheral neutrophils (chemokinesis and chemotaxis toward 10% E. coli; superoxide anion generation after PMA; leukotriene B4 (LTB4) release from whole blood and isolated neutrophtls, before and after different stimuli) were evaluated during an acute exacerbation of asthma, and after 14 – 54 days of treatment with systemic glucocorticosteroids (GCS). During acute exacerbation, superoxide anion generation was higher in asthmatics than in eleven normal subjects (39.2 ± 14.1 vs. 25.2 ± 7.3 nmol, p < 0.05); there was a significant correlation between FEV1 (% of predicted) and neutrophil chemotaxis (r = −0.52, p = 0.04). After treatment, there was no significant change in all neutrophil functions, except for a decrease in neutrophil chemotaxis in subjects who showed an FEV1 increase > 20% after GCS treatment (from 131 ± 18 to 117 ± 21 μm, p = 0.005). Chemokinesis sicantly decreased in all subjects, and the changes significantly correlated with an arbitrary score of the total administered dose of GCS (r = 0.57, p < 0.05). These data suggest that neutrophil activation plays a minor role in asthma, and that treatment with GCS is not able to modify most functions of peripheral neutrophils in asthmatic subjects; chemotaxis seems to be related only to the severity of the asthma and it could reflect the improvement of the disease. PMID:18475647

  1. In vivo chemical and structural analysis of plant cuticular waxes using stimulated Raman scattering microscopy.

    PubMed

    Littlejohn, George R; Mansfield, Jessica C; Parker, David; Lind, Rob; Perfect, Sarah; Seymour, Mark; Smirnoff, Nicholas; Love, John; Moger, Julian

    2015-05-01

    The cuticle is a ubiquitous, predominantly waxy layer on the aerial parts of higher plants that fulfils a number of essential physiological roles, including regulating evapotranspiration, light reflection, and heat tolerance, control of development, and providing an essential barrier between the organism and environmental agents such as chemicals or some pathogens. The structure and composition of the cuticle are closely associated but are typically investigated separately using a combination of structural imaging and biochemical analysis of extracted waxes. Recently, techniques that combine stain-free imaging and biochemical analysis, including Fourier transform infrared spectroscopy microscopy and coherent anti-Stokes Raman spectroscopy microscopy, have been used to investigate the cuticle, but the detection sensitivity is severely limited by the background signals from plant pigments. We present a new method for label-free, in vivo structural and biochemical analysis of plant cuticles based on stimulated Raman scattering (SRS) microscopy. As a proof of principle, we used SRS microscopy to analyze the cuticles from a variety of plants at different times in development. We demonstrate that the SRS virtually eliminates the background interference compared with coherent anti-Stokes Raman spectroscopy imaging and results in label-free, chemically specific confocal images of cuticle architecture with simultaneous characterization of cuticle composition. This innovative use of the SRS spectroscopy may find applications in agrochemical research and development or in studies of wax deposition during leaf development and, as such, represents an important step in the study of higher plant cuticles.

  2. FFTF Plant transition function analysis report

    SciTech Connect

    Lund, D.P.; FFTF Working Group

    1995-09-01

    The document contains the functions, function definitions, function interfaces, function interface definitions, Input Computer Automated Manufacturing Definition (IDEFO) diagrams, and function hierarchy charts that describe what needs to be performed to deactivate FFTF.

  3. The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo

    PubMed Central

    Hostettler, Lola; Grundy, Laura; Käser-Pébernard, Stéphanie; Wicky, Chantal; Schafer, William R.; Glauser, Dominique A.

    2017-01-01

    The Green Fluorescent Protein (GFP) has been tremendously useful in investigating cell architecture, protein localization, and protein function. Recent developments in transgenesis and genome editing methods now enable working with fewer transgene copies and, consequently, with physiological expression levels. However, lower signal intensity might become a limiting factor. The recently developed mNeonGreen protein is a brighter alternative to GFP in vitro. The goal of the present study was to determine how mNeonGreen performs in vivo in Caenorhabditis elegans—a model used extensively for fluorescence imaging in intact animals. We started with a side-by-side comparison between cytoplasmic forms of mNeonGreen and GFP expressed in the intestine, and in different neurons, of adult animals. While both proteins had similar photostability, mNeonGreen was systematically 3–5 times brighter than GFP. mNeonGreen was also used successfully to trace endogenous proteins, and label specific subcellular compartments such as the nucleus or the plasma membrane. To further demonstrate the utility of mNeonGreen, we tested transcriptional reporters for nine genes with unknown expression patterns. While mNeonGreen and GFP reporters gave overall similar expression patterns, low expression tissues were detected only with mNeonGreen. As a whole, our work establishes mNeonGreen as a brighter alternative to GFP for in vivo imaging in a multicellular organism. Furthermore, the present research illustrates the utility of mNeonGreen to tag proteins, mark subcellular regions, and describe new expression patterns, particularly in tissues with low expression. PMID:28108553

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

    PubMed Central

    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

    2011-01-01

    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. PMID:20829246

  5. Effects of cortisol on pregnancy rate and corpus luteum function in heifers: an in vivo study.

    PubMed

    Duong, Hai Thanh; Piotrowska-Tomala, Katarzyna Karolina; Acosta, Tomas Javier; Bah, Mamadou Mousa; Sinderewicz, Emilia; Majewska, Magdalena; Jankowska, Katarzynna; Okuda, Kiyoshi; Skarzynski, Dariusz Jan

    2012-01-01

    To determine whether glucocorticoids affect the function of the bovine corpus luteum (CL) during the estrous cycle and early pregnancy, we examined the effects of exogenous cortisol or reduced endogenous cortisol on the secretion of progesterone (P4) and on pregnancy rate. In preliminary experiments, doses of cortisol and metyrapone (an inhibitor of cortisol synthesis) were established (n=33). Cortisol in effective doses of 10 mg blocked tumor necrosis factor-induced prostaglandin F(2α) secretion as measured by its metabolite (PGFM) concentrations in the blood. Metyrapone in effective doses of 500 mg increased the P4 concentration. Thus, both reagents were then intravaginally applied in the chosen doses daily from Day 15 to 18 after estrus (Day 0) in noninseminated heifers (n=18) or after artificial insemination (n=36). Pregnancy was confirmed by transrectal ultrasonography between Days 28-30 after insemination. Plasma concentrations of P4 were lower in cortisol-treated heifers than in control heifers on Days 17 and 18 of the estrous cycle (P<0.05). However, the interestrus intervals were not different between control and cortisol-treated animals (P>0.05). Moreover, metyrapone increased P4 and prolonged the CL lifespan in comparison to control animals (P<0.05). Interestingly, in inseminated heifers, cortisol increased the pregnancy rate (75%) compared with control animals (58%), whereas metyrapone reduced the pregnancy rate to 16.7% (P<0.05). The overall results suggest that cortisol, depending on the physiological status of heifers (pregnant vs. nonpregnant), modulates CL function by influencing P4 secretion. Cortisol may have a positive influence on CL function during early pregnancy, leading to support of embryo implantation and resulting in higher rates of pregnancy in heifers.

  6. Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90.

    PubMed Central

    Blagosklonny, M V; Toretsky, J; Bohen, S; Neckers, L

    1996-01-01

    The p53 mutant, 143ala, was translated in vitro in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE). In RRL, p53-143ala protein of both mutant and wild-type conformation, as detected immunologically with conformation-specific antibodies, was translated. The chaperone protein HSP90, present in RRL, was found to coprecipitate only with the mutated conformation of p53. Geldanamycin, shown previously to bind to HSP90 and destabilize its association with other proteins, decreased the amount of immunologically detectable mutated p53 and increased the amount of detectable wild-type protein, without affecting the total translation of p53. When translated in WGE, known to contain functionally deficient HSP90, p53-143ala produced p53 protein, which was not recognized by a mutated conformation-specific antibody. In contrast, the synthesis of conformationally detectable wild-type p53 in this system was not compromised. Reconstitution of HSP90 function in WGE permitted synthesis of conformationally detectable mutated p53, and this was abrogated by geldanamycin. Finally, when p53-143ala was stably tansfected into yeast engineered to be defective for HSP90 function, conformational recognition of mutated p53 was impaired. When stable transfectants of p53-143ala were prepared in yeast expressing wild-type HSP90, conformational recognition of mutated p53 was antagonized by macbecin I, a geldanamycin analog also known to bind HSP90. Taken together, these data demonstrate a role for HSP90 in the achievement and/or stabilization of the mutated conformation of p53-143ala. Furthermore, we show that the mutated conformation of p53 can be pharmacologically antagonized by drugs targeting HSP90. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8710879

  7. P-31-NMR analysis of in vivo metabolic events in lymphoma during RIT

    SciTech Connect

    Adams, D.A.; DeNardo, G.L.; DeNardo, S.J.

    1985-05-01

    Radioimmunotherapy (RIT) is potentially a powerful method for treatment of cancer. The efficacy of radioimmunopharmaceuticals on target tissue were measured serially, in vivo, in the human non-Hodgkin's lymphoma (RAJI tum