Science.gov

Sample records for vivo functional analysis

  1. In vivo analysis of functional domains from villin and gelsolin

    PubMed Central

    1992-01-01

    Transfected CV1 cells were used to compare the in vivo effects of various domains of villin and gelsolin. These two homologous actin modulating proteins both contain a duplicated severin-like sequence. Villin has in addition a carboxy-terminal domain, the headpiece, which accounts for its bundling activity. The effects of the villin-deleted mutants were compared with those of native villin. Our results show that essential domains of villin required to induce the growth of microvilli and F-actin redistribution are present in the first half of the core and in the headpiece. We also show that the second half of the villin core cannot be exchanged by its homolog in gelsolin. When expressed at high levels of CV1 cells, full length gelsolin completely disrupted stress fibers without change of the cell shape. Addition of the villin headpiece to gelsolin had no effect on the phenotype induced by gelsolin alone. Expression of the first half of gelsolin induced similar modifications as capping proteins and rapid cell mortality; this deleterious effect on the cell structure was also observed when the headpiece was linked to the first half of gelsolin. In cells expressing the second half of gelsolin, a dotted F-actin staining was often seen. Moreover elongated dorsal F-actin structures were observed when the headpiece was linked to the second gelsolin domain. These studies illustrate the patent in vivo severing activity of gelsolin as well as the distinct functional properties of villin core in contrast to gelsolin. PMID:1310994

  2. 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 ?-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. PMID:21074529

  3. Methods for Ex Vivo Analysis of Immune Cell Function from the Central Nervous System.

    PubMed

    Turner, Darryl G; Leech, Melanie D; O'Connor, Richard A; Anderton, Stephen M

    2016-01-01

    Experimental autoimmune encephalomyelitis (EAE) is an animal model commonly used to investigate the inflammatory response in organ-specific autoimmunity and a model of the early immune responses of multiple sclerosis.This protocol outlines the methods used for the processing of peripheral immune tissues, the spleen and draining lymph nodes, as well as the site of inflammation, the central nervous system (CNS), for analyzing immune cell phenotype and function during murine EAE. PMID:25863784

  4. In vivo functional analysis of the Drosophila melanogaster nicotinic acetylcholine receptor D?6 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, D?6. Reciprocal chimeric subunits were created from D?6 and D?7, a subunit that does not respond to spinosad. Using the in vivo system, the D?6/D?7 chimeric subunits were tested for their capacity to respond to spinosad. Only the subunits containing the C-terminal region of D?6 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 D?6(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. PMID:25747007

  5. Ex Vivo ERG analysis of photoreceptors using an In Vivo ERG system

    PubMed Central

    Vinberg, Frans; Kolesnikov, Alexander V.; Kefalov, Vladimir J.

    2014-01-01

    The Function of the retina and effects of drugs on it can be assessed by recording transretinal voltage across isolated retina that is perfused with physiological medium. However, building ex vivo ERG apparatus requires substantial amount of time, resources and expertise. Here we adapted a commercial in vivo ERG system for transretinal ERG recordings from rod and cone photoreceptors and compared rod and cone signalling between ex vivo and in vivo environments. We found that the rod and cone a- and b-waves recorded with the transretinal ERG adapter and a standard in vivo ERG system are comparable to those obtained from live anesthetized animals. However, ex vivo responses are somewhat slower and their oscillatory potentials are suppressed as compared to those recorded in vivo. We found that rod amplification constant (A) was comparable between ex vivo and in vivo conditions, ?10 - 30 s-2 depending on the choice of response normalization. We estimate that the A in cones is between 3 and 6 s-2 in ex vivo conditions and by assuming equal A in vivo we arrive to light funnelling factor of 3 for cones in the mouse retina. The ex vivo ERG adapter provides a simple and affordable alternative to designing a custom-built transretinal recordings setup for the study of photoreceptors. Our results provide a roadmap to the rigorous quantitative analysis of rod and cone responses made possible with such a system. PMID:24959652

  6. Overcoming the heterologous bias: An in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata

    SciTech Connect

    Puri, Nidhi; Manoharlal, Raman; Sharma, Monika; Sanglard, Dominique; Prasad, Rajendra

    2011-01-07

    Research highlights: {yields} First report to demonstrate an in vivo expression system of an ABC multidrug transporter CgCdr1p of C. glabrata. {yields} First report on the structure and functional characterization of CgCdr1p. {yields} Functional conservation of divergent but typical residues of CgCdr1p. {yields} CgCdr1p elicits promiscuity towards substrates and has a large drug binding pocket with overlapping specificities. -- Abstract: We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the arte-factual concerns encountered in using heterologous systems are totally excluded.

  7. Phenotypical Analysis of Atypical PKCs In Vivo Function Display a Compensatory System at Mouse Embryonic Day 7.5

    PubMed Central

    Seidl, Sebastian; Braun, Ursula; Roos, Norbert; Li, Shaohua; Lüdtke, Timo H.-W.

    2013-01-01

    Background The atypical protein kinases C (PKC) isoforms ?/? and ? play crucial roles in many cellular processes including development, cell proliferation, differentiation and cell survival. Possible redundancy between the two isoforms has always been an issue since most biochemical tools do not differentiate between the two proteins. Thus, much effort has been made during the last decades to characterize the functions of aPKCs using gene targeting approaches and depletion studies. However, little is known about the specific roles of each isoform in mouse development. Methodology/Principal Findings To evaluate the importance of PKC? in mouse development we designed PKC? deletion mutants using the gene targeting approach. We show that the deletion of PKC?, results in a reduced size of the amniotic cavity at E7.5 and impaired growth of the embryo at E8.5 with subsequent absorption of the embryo. Our data also indicate an impaired localization of ZO-1 and disorganized structure of the epithelial tissue in the embryo. Importantly, using electron microscopy, embryoid body formation and immunofluorescence analysis, we found, that in the absence of PKC?, tight junctions and apico-basal polarity were still established. Finally, our study points to a non-redundant PKC? function at E9.5, since expression of PKC? is able to rescue the E7.5 phenotype, but could not prevent embryonic lethality at a later time-point (E9.5). Conclusion Our data show that PKC? is crucial for mouse embryogenesis but is dispensable for the establishment of polarity and tight junction formation. We present a compensatory function of PKC? at E7.5, rescuing the phenotype. Furthermore, this study indicates at least one specific, yet unknown, PKC? function that cannot be compensated by the overexpression of PKC? at E9.5. PMID:23690951

  8. Targeted Gene Deletion and In Vivo Analysis of Putative Virulence Gene Function in the Pathogenic Dermatophyte Arthroderma benhamiae?

    PubMed Central

    Grumbt, Maria; Defaweux, Valérie; Mignon, Bernard; Monod, Michel; Burmester, Anke; Wöstemeyer, Johannes; Staib, Peter

    2011-01-01

    Dermatophytes cause the majority of superficial mycoses in humans and animals. However, little is known about the pathogenicity of this specialized group of filamentous fungi, for which molecular research has been limited thus far. During experimental infection of guinea pigs by the human pathogenic dermatophyte Arthroderma benhamiae, we recently detected the activation of the fungal gene encoding malate synthase AcuE, a key enzyme of the glyoxylate cycle. By the establishment of the first genetic system for A. benhamiae, specific ?acuE mutants were constructed in a wild-type strain and, in addition, in a derivative in which we inactivated the nonhomologous end-joining pathway by deletion of the A. benhamiae KU70 gene. The absence of AbenKU70 resulted in an increased frequency of the targeted insertion of linear DNA by homologous recombination, without notably altering the monitored in vitro growth abilities of the fungus or its virulence in a guinea pig infection model. Phenotypic analyses of ?acuE mutants and complemented strains depicted that malate synthase is required for the growth of A. benhamiae on lipids, major constituents of the skin. However, mutant analysis did not reveal a pathogenic role of the A. benhamiae enzyme in guinea pig dermatophytosis or during epidermal invasion of the fungus in an in vitro model of reconstituted human epidermis. The presented efficient system for targeted genetic manipulation in A. benhamiae, paired with the analyzed infection models, will advance the functional characterization of putative virulence determinants in medically important dermatophytes. PMID:21478433

  9. Structural characterization of the medfly hsp83 gene and functional analysis of its proximal promoter region in vivo by germ-line transformation.

    PubMed

    Theodoraki, Maria; Tatari, Marianthi; Chrysanthis, George; Zacharopoulou, Antigone; Mintzas, Anastassios C

    2008-01-01

    In order to define the regulatory elements responsible for the expression of the medfly hsp83 (Cchsp83) gene, we determined the sequence of a genomic region of the gene that included 3,536 bp upstream of the transcription initiation site, the first untranslated exon of 144 bp, a 275-bp intron, and 516 bp of the second coding exon. Structural analysis of the 5' flanking region revealed the presence of a typical TATA box, 28 bp upstream of the transcription start site, and seven putative heat shock elements (HSEs) further upstream. The 5' untranslated region of the Cchsp83 mRNA was found to contain extensive secondary structure in the first 126 nucleotides. We carried out deletion functional analysis of the proximal promoter region (-380/+139) in vivo by germ line transformation using the lacZ as a reporter gene. We found that sequences in the -380/-86 region are essential for the constitutive expression of the Cchsp83 gene. Under normal conditions, the -380/+139 region was able to drive significant levels of transgene expression in all developmental stages of the medfly as well as in the ovaries and testis. In most stages, the temporal expression pattern of the reporter gene was similar to the respective pattern of the endogenous Cchsp83 gene. Although the -380/+139 promoter region contained two putative HSEs, it was found unable to confer any heat-induced expression in the reporter gene. PMID:18064699

  10. 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 ...

  11. In Vivo Imaging of Tissue Physiological Function

    Cancer.gov

    The National Cancer Institute's Radiation Biology Branch is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize methods for in vivo imaging.

  12. Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

    PubMed Central

    Vinberg, Frans; Kefalov, Vladimir

    2015-01-01

    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. PMID:25992809

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

    PubMed

    Vinberg, Frans; Kefalov, Vladimir

    2015-01-01

    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. PMID:25992809

  14. Assessment of Glial Function in the In Vivo Retina

    PubMed Central

    Srienc, Anja I.; Kornfield, Tess E.; Mishra, Anusha; Burian, Michael A.; Newman, Eric A.

    2013-01-01

    Glial cells, traditionally viewed as passive elements in the CNS, are now known to have many essential functions. Many of these functions have been revealed by work on retinal glial cells. This work has been conducted almost exclusively on ex vivo preparations and it is essential that retinal glial cell functions be characterized in vivo as well. To this end, we describe an in vivo rat preparation to assess the functions of retinal glial cells. The retina of anesthetized, paralyzed rats is viewed with confocal microscopy and laser speckle flowmetry to monitor glial cell responses and retinal blood flow. Retinal glial cells are labeled with the Ca2+ indicator dye Oregon Green 488 BAPTA-1 and the caged Ca2+ compound NP-EGTA by injection of the compounds into the vitreous humor. Glial cells are stimulated by photolysis of caged Ca2+ and the activation state of the cells assessed by monitoring Ca2+ indicator dye fluorescence. We find that, as in the ex vivo retina, retinal glial cells in vivo generate both spontaneous and evoked intercellular Ca2+ waves. We also find that stimulation of glial cells leads to the dilation of neighboring retinal arterioles, supporting the hypothesis that glial cells regulate blood flow in the retina. This in vivo preparation holds great promise for assessing glial cell function in the healthy and pathological retina. PMID:22144328

  15. Function of tubulin binding proteins in vivo.

    PubMed Central

    Fleming, J A; Vega, L R; Solomon, F

    2000-01-01

    Overexpression of the beta-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant alpha-tubulin, tub1-724, that produces unstable heterodimer. Here we use RBL2 overexpression to identify mutations in other genes that affect formation or stability of heterodimer. This approach identifies four genes-CIN1, CIN2, CIN4, and PAC2-as affecting heterodimer formation in vivo. The vertebrate homologues of two of these gene products-Cin1p/cofactor D and Pac2p/cofactor E-can catalyze exchange of tubulin polypeptides into preexisting heterodimer in vitro. Previous work suggests that both Cin2p or Cin4p act in concert with Cin1p in yeast, but no role for vertebrate homologues of either has been reported in the in vitro reaction. Results presented here demonstrate that these proteins can promote heterodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant cells causes microtubule disassembly and enhanced formation of Rbl2p-beta-tubulin complex, as it does in the alpha-tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant alpha-tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential under conditions where the levels of dissociated tubulin polypeptides increase. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of free beta-tubulin. PMID:10978276

  16. In vivo tests of thermodynamic models of transcription repressor function.

    PubMed

    Tungtur, Sudheer; Skinner, Harlyn; Zhan, Hongli; Swint-Kruse, Liskin; Beckett, Dorothy

    2011-11-01

    One emphasis of the Gibbs Conference on Biothermodynamics is the value of thermodynamic measurements for understanding behaviors of biological systems. In this study, the correlation between thermodynamic measurements of in vitro DNA binding affinity with in vivo transcription repression was investigated for two transcription repressors. In the first system, which comprised an engineered LacI/GalR homolog, mutational changes altered the equilibrium constant for binding DNA. Changes correlated with altered repression, but estimates of in vivo repressor concentration suggest a ?25-fold discrepancy with in vitro conditions. In the second system, changes in ligand binding to BirA altered dimerization and subsequent DNA occupancy. Again, these changes correlate with altered in vivo repression, but comparison with in vitro measurements reveals a ~10-fold discrepancy. Further analysis of each system suggests that the observed discrepancies between in vitro and in vivo results reflect the contributions of additional equilibria to the transcription repression process. PMID:21715082

  17. Immunolocalization and in vivo Functional Analysis by RNAi of the Aedes Kinin Receptor in Female Mosquitoes of Aedes aegypti (L.) (Diptera, Culicidae) 

    E-print Network

    Kersch, Cymon

    2012-02-14

    , followed by measurement of in vivo urine excretion post blood feeding in a precision humidity chamber. Transcript and protein knockdown were confirmed by qPCR and immunohistochemistry, respectively. Results indicate widespread expression of the Aedes kinin...

  18. 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. PMID:26001073

  19. 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

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

    PubMed Central

    Radbruch, Helena; Bremer, Daniel; Mothes, Ronja; Günther, Robert; Rinnenthal, Jan Leo; Pohlan, Julian; Ulbricht, Carolin; Hauser, Anja E.; Niesner, Raluca

    2015-01-01

    The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice. PMID:26006244

  1. Development of functional connections between thalamic fibres and the visual cortex of the wallaby revealed by current source density analysis in vivo.

    PubMed

    Pearce, A R; James, A C; Mark, R F

    2000-03-20

    Functional development of thalamic input to the cortex in anaesthetised wallaby pouch young between postnatal day 25 (P25) and P153 has been studied by electrical stimulation of the optic nerve, current source density (CSD) analysis, and histologic identification of recording sites. Conduction in the optic nerve was recorded prior to P39, by which time responses from the superior colliculus appeared. No evoked potential of cortical origin was recorded until P46, even though thalamic fibres grew into the cortical plate from P15. The first cortical synaptic responses were recorded at the margin of the subplate and the developing cortical plate, where cells that later comprise the adult layer 6 settle. At about P66, an additional short-latency, superficial response appeared, coinciding with the formation of layer 4. The deep response was retained in layer 6. Evoked activity in the presumed layer 4 was found progressively deeper in the cortex over the next few weeks, which would be expected from the addition of layer 3 above it. By P113, a new sink was added superficial in the cortex. Thalamocortical connections follow the same deep-to-superficial order in development as the cellular layers of the cortex. PMID:10713572

  2. In vivo compartmental analysis of leukocytes in mouse lungs.

    PubMed

    Patel, Brijesh V; Tatham, Kate C; Wilson, Michael R; O'Dea, Kieran P; Takata, Masao

    2015-10-01

    The lung has a unique structure consisting of three functionally different compartments (alveolar, interstitial, and vascular) situated in an extreme proximity. Current methods to localize lung leukocytes using bronchoalveolar lavage and/or lung perfusion have significant limitations for determination of location and phenotype of leukocytes. Here we present a novel method using in vivo antibody labeling to enable accurate compartmental localization/quantification and phenotyping of mouse lung leukocytes. Anesthetized C57BL/6 mice received combined in vivo intravenous and intratracheal labeling with fluorophore-conjugated anti-CD45 antibodies, and lung single-cell suspensions were analyzed by flow cytometry. The combined in vivo intravenous and intratracheal CD45 labeling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung. In naive mice, the alveolar compartment consisted predominantly of resident alveolar macrophages. The interstitial compartment, gated by events negative for both intratracheal and intravenous CD45 staining, showed two conventional dendritic cell populations, as well as a Ly6C(lo) monocyte population. Expression levels of MHCII on these interstitial monocytes were much higher than on the vascular Ly6C(lo) monocyte populations. In mice exposed to acid aspiration-induced lung injury, this protocol also clearly distinguished the three lung compartments showing the dynamic trafficking of neutrophils and exudative monocytes across the lung compartments during inflammation and resolution. This simple in vivo dual-labeling technique substantially increases the accuracy and depth of lung flow cytometric analysis, facilitates a more comprehensive examination of lung leukocyte pools, and enables the investigation of previously poorly defined "interstitial" leukocyte populations during models of inflammatory lung diseases. PMID:26254421

  3. Physiological functions of protein kinase D in vivo.

    PubMed

    Ellwanger, Kornelia; Hausser, Angelika

    2013-02-01

    The cellular functions of the serine/threonine protein kinase D (PKD) have been extensively studied within the last decade and distinct roles such as fission of vesicles at the Golgi compartment, coordination of cell migration and invasion, and regulation of gene transcription have been correlated with this kinase family. Here, we highlight the current state of in vivo studies on PKD function with a focus on animal models and discuss the molecular basis of the observed phenotypic characteristics associated with this kinase family. PMID:23288632

  4. In vivo investigation of cilia structure and function using Xenopus

    PubMed Central

    Brooks, Eric R.; Wallingford, John B.

    2015-01-01

    Cilia are key organelles in development and homeostasis. The ever-expanding complement of cilia associated proteins necessitates rapid and tractable models for in vivo functional investigation. Xenopus laevis provides an attractive model for such studies, having multiple ciliated populations, including primary and multiciliated tissues. The rapid external development of Xenopus and the large cells make it an especially excellent platform for imaging studies. Here we present embryological and cell-biological methods for the investigation of cilia structure and function in Xenopus laevis, with a focus on quantitative live and fixed imaging. PMID:25837389

  5. Functional Beta2-Integrins Restrict Skin Inflammation In Vivo.

    PubMed

    Savinko, Terhi S; Morrison, Vicky L; Uotila, Liisa M; Wolff, C Henrik J; Alenius, Harri T; Fagerholm, Susanna C

    2015-09-01

    Beta2-integrins and the important integrin regulator kindlin-3 are essential for leukocyte trafficking, but the role of beta2-integrins in regulating inflammation is still incompletely understood. Here, we have investigated skin inflammation in a mouse model where the kindlin-3 binding site in the beta2-integrin has been mutated (TTT/AAA-beta2-integrin knock-in), leading to expressed but dysfunctional integrins. We show that, surprisingly, neutrophil trafficking into the inflamed skin in a contact hypersensitivity model is normal in these mice, although trafficking of T cells and eosinophils into the skin is reduced. Instead, expression of dysfunctional integrins leads to increased mast cell and dendritic cell numbers in the skin, increased inflammatory cytokine production in the inflamed skin in vivo, and in mast cells in vitro. Furthermore, expression of dysfunctional integrins leads to increased dendritic cell activation and migration to lymph nodes and increased Th1 responses in vivo. Therefore, the kindlin-3/integrin interaction is important for trafficking of T cells and eosinophils but not absolutely required for neutrophil trafficking into the inflamed skin. Functional beta2-integrins also have a major role in restricting the immune response in the inflamed skin and lymph nodes in vivo, likely through effects on mast cell and dendritic cell numbers and activation. PMID:25918984

  6. Bacterial ApbC protein has two biochemical activities that are required for in vivo function.

    PubMed

    Boyd, Jeffrey M; Sondelski, Jamie L; Downs, Diana M

    2009-01-01

    The ApbC protein has been shown previously to bind and rapidly transfer iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J., Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47, 8195-8202. This study utilized both in vivo and in vitro assays to examine the function of variant ApbC proteins. The in vivo assays assessed the ability of ApbC proteins to function in pathways with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S] cluster, and transfer [Fe-S] cluster. This study details the first kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of "deviant" Walker A proteins. Moreover, this study details the first functional analysis of mutant variants of the ever expanding family of ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that ApbC protein needs ATPase activity and the ability to bind and rapidly transfer [Fe-S] clusters for in vivo function. PMID:19001370

  7. Bacterial ApbC Protein Has Two Biochemical Activities That Are Required for in Vivo Function*

    PubMed Central

    Boyd, Jeffrey M.; Sondelski, Jamie L.; Downs, Diana M.

    2009-01-01

    The ApbC protein has been shown previously to bind and rapidly transfer iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J., Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47, 8195–8202. This study utilized both in vivo and in vitro assays to examine the function of variant ApbC proteins. The in vivo assays assessed the ability of ApbC proteins to function in pathways with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S] cluster, and transfer [Fe-S] cluster. This study details the first kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of “deviant” Walker A proteins. Moreover, this study details the first functional analysis of mutant variants of the ever expanding family of ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that ApbC protein needs ATPase activity and the ability to bind and rapidly transfer [Fe-S] clusters for in vivo function. PMID:19001370

  8. Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice

    PubMed Central

    Reggi, Serena; Tchou-Wong, Kam-Meng; Rom, William N.; Busconi, Matteo; Fogher, Corrado

    2016-01-01

    Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe’s disease. To investigate whether we could generate a functional recombinant human GAA enzyme (tobrhGAA) in tobacco seeds for future enzyme replacement therapy, we subcloned the human GAA cDNA into the plant expression plasmid-pBI101 under the control of the soybean ?-conglycinin seed-specific promoter and biochemically analyzed the tobrhGAA. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation–phosphorylation and processing. We found the tobrhGAA to be enzymatically active was readily taken up by GSDII fibroblasts and in white blood cells from whole blood to reverse the defect. The tobrhGAA corrected the enzyme defect in tissues at 7 days after a single dose following intraperitoneal (IP) administration in GAA knockout (GAA?/?) mice. Additionally, we could purify the tobrhGAA since it bound tightly to the matrix of Sephadex G100 and can be eluted by competition with maltose. These data demonstrate indirectly that the tobrhGAA is fully functional, predominantly proteolytically cleaved and contains the minimal phosphorylation and mannose-6-phosphate residues essential for biological activity. PMID:23907679

  9. Comparison of three formal methods used to estimate the functional axis of rotation: an extensive in-vivo analysis performed on the knee joint.

    PubMed

    Colle, Francesca; Lopomo, Nicola; Visani, Andrea; Zaffagnini, Stefano; Marcacci, Maurilio

    2016-04-01

    Estimating the main axis of rotation (AoR) of a human joint represents an important issue in biomechanics. This study compared three formal methods used to estimate functional AoR, namely a cylindrical fitting method, a mean helical axis transformation, and a symmetrical axis approach. These methods were tested on 106 subjects undergoing navigated total knee arthroplasty. AoR orientation in 3D and in the frontal and coronal planes provided by each method was compared to the transepicondylar axis direction. Although all the methods resulted effective, significant differences were identified among them, relatively to the orientation in 3D and in the frontal plane projection. This was probably due to the presence of secondary rotations during the first degrees of knee flexion. PMID:26207419

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

    PubMed Central

    Attridge, Kesley; Walker, Lucy S K

    2014-01-01

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

  11. 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…

  12. Mapping actin surfaces required for functional interactions in vivo

    PubMed Central

    1994-01-01

    An in vivo strategy to identify amino acids of actin required for functional interactions with actin-binding proteins was developed. This approach is based on the assumption that an actin mutation that specifically impairs the interaction with an actin-binding protein will cause a phenotype similar to a null mutation in the gene that encodes the actin-binding protein. 21 actin mutations were analyzed in budding yeast, and specific regions of actin subdomain 1 were implicated in the interaction with fimbrin, an actin filament-bundling protein. Mutations in this actin subdomain were shown to be, like a null allele of the yeast fimbrin gene (SAC6), lethal in combination with null mutations in the ABP1 and SLA2 genes, and viable in combination with a null mutation in the SLA1 gene. Biochemical experiments with act1-120 actin (E99A, E100A) verified a defect in the fimbrin-actin interaction. Genetic interactions between mutant alleles of the yeast actin gene and null alleles of the SAC6, ABP1, SLA1, and SLA2 genes also demonstrated that the effects of the 21 actin mutations are diverse and allowed four out of seven pseudo-wild-type actin alleles to be distinguished from the wild-type gene for the first time, providing evidence for functional redundancy between different surfaces of actin. PMID:8034743

  13. In vivo predictive dissolution: transport analysis of the CO2 , bicarbonate in vivo buffer system.

    PubMed

    Krieg, Brian J; Taghavi, Seyed Mohammad; Amidon, Gordon L; Amidon, Gregory E

    2014-11-01

    Development of an oral in vivo predictive dissolution medium for acid drugs with a pKa in the physiological range (e.g., Biopharmaceutics Classification System Class IIa) requires transport analysis of the complex in vivo CO2 /bicarbonate buffering system. In this report, we analyze this buffer system using hydrodynamically defined rotating disk dissolution. Transport analysis of drug flux was predicted using the film model approach of Mooney et al based on equilibrium assumptions as well as accounting for the slow hydration reaction, CO2 + H2 O ? H2 CO3 . The accuracy of the models was compared with experimentally determined results using the rotating disk dissolution of ibuprofen, indomethacin, and ketoprofen. The equilibrium and slow hydration reaction rate models predict significantly different dissolution rates. The experimental results are more accurately predicted by accounting for the slow hydration reaction under a variety of pH and hydrodynamic conditions. Although the complex bicarbonate buffering system requires further consideration given its dynamic nature in vivo, a simplifying irreversible reaction (IRR) transport analysis accurately predicts in vitro rotating disk dissolution rates of several carboxylic acid drugs. This IRR transport model provides further insight into bicarbonate buffer and can be useful in developing more physiologically relevant buffer systems for dissolution testing. PMID:25212721

  14. 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.

  15. An in vivo assay of synaptic function mediating human cognition.

    PubMed

    Moran, Rosalyn J; Symmonds, Mkael; Stephan, Klaas E; Friston, Karl J; Dolan, Raymond J

    2011-08-01

    The contribution of dopamine to working memory has been studied extensively [1-3]. Here, we exploited its well characterized effects [1-3] to validate a novel human in vivo assay of ongoing synaptic [4, 5] processing. We obtained magnetoencephalographic (MEG) measurements from subjects performing a working memory (WM) task during a within-subject, placebo-controlled, pharmacological (dopaminergic) challenge. By applying dynamic causal modeling (DCM), a Bayesian technique for neuronal system identification [6], to MEG signals from prefrontal cortex, we demonstrate that it is possible to infer synaptic signaling by specific ion channels in behaving humans. Dopamine-induced enhancement of WM performance was accompanied by significant changes in MEG signal power, and a DCM assay disclosed related changes in synaptic signaling. By estimating the contribution of ionotropic receptors (AMPA, NMDA, and GABA(A)) to the observed spectral response, we demonstrate changes in their function commensurate with the synaptic effects of dopamine. The validity of our model is reinforced by a striking quantitative effect on NMDA and AMPA receptor signaling that predicted behavioral improvement over subjects. Our results provide a proof-of-principle demonstration of a novel framework for inferring, noninvasively, neuromodulatory influences on ion channel signaling via specific ionotropic receptors, providing a window on the hidden synaptic events mediating discrete psychological processes in humans. PMID:21802302

  16. 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

  17. 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. PMID:26440436

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

    PubMed Central

    2010-01-01

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

  19. 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…

  20. A subject-specific framework for in vivo myeloarchitectonic analysis using high resolution quantitative MRI.

    PubMed

    Waehnert, Miriam D; Dinse, Juliane; Schäfer, Andreas; Geyer, Stefan; Bazin, Pierre-Louis; Turner, Robert; Tardif, Christine Lucas

    2016-01-15

    Structural magnetic resonance imaging can now resolve laminar features within the cerebral cortex in vivo. A variety of intracortical contrasts have been used to study the cortical myeloarchitecture with the purpose of mapping cortical areas in individual subjects. In this article, we first briefly review recent advances in MRI analysis of cortical microstructure to portray the potential and limitations of the current state-of-the-art. We then present an integrated framework for the analysis of intracortical structure, composed of novel image processing tools designed for high resolution cortical images. The main features of our framework are the segmentation of quantitative T1 maps to delineate the cortical boundaries (Bazin et al., 2014), and the use of an equivolume layering model to define an intracortical coordinate system that follows the anatomical layers of the cortex (Waehnert et al., 2014). We evaluate the framework with 150?m isotropic post mortem T2(?)-weighted images and 0.5mm isotropic in vivo T1 maps, a quantitative index of myelin content. We study the laminar structure of the primary visual cortex (Brodmann area 17) in the post mortem and in vivo data, as well as the central sulcus region in vivo, in particular Brodmann areas 1, 3b and 4. We also investigate the impact of the layering models on the relationship between T1 and cortical curvature. Our experiments demonstrate that the equivolume intracortical surfaces and transcortical profiles best reflect the laminar structure of the cortex in areas of curvature in comparison to the state-of-the-art equidistant and Laplace implementations. This framework generates a subject specific intracortical coordinate system, the basis for subsequent architectonic analyses of the cortex. Any structural or functional contrast co-registered to the T1 maps, used to segment the cortex, can be sampled on the curved grid for analysis. This work represents an important step towards in vivo structural brain mapping of individual subjects. PMID:26455795

  1. Yeast Functional Analysis Report Functional analysis of the Saccharomyces

    E-print Network

    Alexandraki, Despina

    Yeast Functional Analysis Report Functional analysis of the Saccharomyces cerevisiae YFR021w/YGR223 mitochondria-related functions for all three ORFs. Two-hybrid screens of a yeast genomic library identified. Under most conditions examined, the effects of the single- and double-ORF deletions indicated that YFR

  2. 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.

  3. 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.

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

    PubMed

    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

  5. A Numerical Analysis Model for Interpretation of Flow Cytometric Studies of Ex Vivo Phagocytosis

    PubMed Central

    Strom, Ted S.; Anur, Praveen; Prislovsky, Amanda

    2011-01-01

    The study of ex vivo phagocytosis via flow cytometry requires that one distinguish experimentally between uptake and adsorption of fluorescently labeled targets by phagocytes. Removal of the latter quantity from the analysis is the most common means of analyzing such data. Because the probability of phagocytosis is a function of the probability of adsorption, and because partially quenched fluorescence after uptake often overlaps with that of negative controls, this approach is suboptimal at best. Here, we describe a numerical analysis model which overcomes these limitations. We posit that the random adsorption of targets to macrophages, and subsequent phagocytosis, is a function of three parameters: the ratio of targets to macrophages (m), the mean fluorescence intensity imparted to the phagocyte by the internalized target (alpha), and the probability of phagocytosis per adsorbed target (p). The potential values of these parameters define a parameter space and their values at any point in parameter space can be used to predict the fraction of adsorption(+) and [adsorption(?), phagocytosis(+)] cells that might be observed experimentally. By systematically evaluating the points in parameter space for the latter two values and comparing them to experimental data, the model arrives at sets of parameter values that optimally predict such data. Using activated THP-1 cells as macrophages and platelets as targets, we validate the model by demonstrating that it can distinguish between the effects of experimental changes in m, alpha, and p. Finally, we use the model to demonstrate that platelets from a congenitally thrombocytopenic WAS patient show an increased probability of ex vivo phagocytosis. This finding correlates with other evidence that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. Our numerical analysis method represents a useful and innovative approach to multivariate analysis. PMID:22073181

  6. 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...

  7. 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.

  8. Longitudinal Functional Data Analysis

    PubMed Central

    Park, So Young; Staicu, Ana-Maria

    2015-01-01

    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.

  9. 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. PMID:22261097

  10. 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,…

  11. 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,...

  12. 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...

  13. 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...

  14. 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. PMID:19904577

  15. A Multifunctional Turnip Crinkle Virus Replication Enhancer Revealed by in vivo Functional SELEX

    E-print Network

    Simon, Anne

    A Multifunctional Turnip Crinkle Virus Replication Enhancer Revealed by in vivo Functional SELEX College Park College Park, MD 20742, USA The motif1-hairpin (M1H), located on (2)-strands of Turnip, Turnip Crinkle Virus; SELEX, systematic evolution of ligands by exponential enrichment; M1H, motif1

  16. ?5 and ?v integrins cooperate to regulate vascular smooth muscle and neural crest functions in vivo

    E-print Network

    Turner, Christopher J.

    The RGD-binding ?5 and ?v integrins have been shown to be key regulators of vascular smooth muscle cell (vSMC) function in vitro. However, their role on vSMCs during vascular development in vivo remains unclear. To address ...

  17. 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

  18. A Dynamic Real Time In Vivo and Static Ex Vivo Analysis of Granulomonocytic Cell Migration in the Collagen-Induced Arthritis Model

    PubMed Central

    Byrne, Ruth; Rath, Eva; Hladik, Anastasiya; Niederreiter, Birgit; Bonelli, Michael; Frantal, Sophie; Smolen, Josef S.; Scheinecker, Clemens

    2012-01-01

    Neutrophilic granulocytes and monocytes (granulomonocytic cells; GMC) drive the inflammatory process at the earliest stages of rheumatoid arthritis (RA). The migratory behavior and functional properties of GMC within the synovial tissue are, however, only incompletely characterized. Here we have analyzed GMC in the murine collagen-induced arthritis (CIA) model of RA using multi-photon real time in vivo microscopy together with ex vivo analysis of GMC in tissue sections. GMC were abundant as soon as clinical arthritis was apparent. GMC were motile and migrated randomly through the synovial tissue. In addition, we observed the frequent formation of cell clusters consisting of both neutrophilic granulocytes and monocytes that actively contributed to the inflammatory process of arthritis. Treatment of animals with a single dose of prednisolone reduced the mean velocity of cell migration and diminished the overall immigration of GMC. In summary, our study shows that the combined application of real time in vivo microscopy together with elaborate static post-mortem analysis of GMC enables the description of dynamic migratory characteristics of GMC together with their precise location in a complex anatomical environment. Moreover, this approach is sensitive enough to detect subtle therapeutic effects within a very short period of time. PMID:22529989

  19. Defining Uremic Arterial Functional Abnormalities in Patients Recently Started on Haemodialysis: Combined In Vivo and Ex Vivo Assessment

    PubMed Central

    Abushufa, Adil M.; Eldehni, Mohamed T.; Odudu, Aghogho; Evans, Philip D.; O?Sullivan, Saoirse E.; McIntyre, Chris W.

    2014-01-01

    Endothelial dysfunction is a key initiating event in vascular disease in chronic kidney disease (CKD) patients and haemodialysis (HD) patients exhibit significant vascular abnormalities. To understand this further, we examined how ex vivo intrinsic function in isolated arteries correlates with in vivo assessments of cardiovascular status in HD patients. Abdominal fat biopsies were obtained from 11 HD patients and 26 non-uremic controls. Subcutaneous arteries were dissected and mounted on a wire myograph, and cumulative concentration-response curves to noradrenalin, endothelin-1, a thromboxane A2 agonist (U46619), angiotensin II, vasopressin, bradykinin (BK), acetylcholine (ACh) and sodium nitroprusside (SNP) were constructed. Pulse wave velocity and blood pressure were measured in HD patients. Enhanced (P<0.05?0.0001) maximal contractile responses (Rmax) to all spasmogens (particularly vasopressin) were observed in arteries from HD patients compared to controls, and this effect was more pronounced in arteries with an internal diameter>600 µm. The potency (pEC50) of U46619 (P<0.01) and vasopressin (P<0.001) was also increased in arteries>600 µm of HD patients. The maximal relaxant response to the endothelium-dependent dilators ACh and BK were lower in HD patients (P<0.01-P<0.0001) (worse for ACh than BK); however the endothelium-independent dilator SNP was similar in both groups. PWV was significantly correlated with the vasoconstrictor response to vasopressin (P?=?0.042) in HD patients. HD patients are primed for hypertension and end organ demand ischaemia by a highly sensitised pressor response. The failure of arterial relaxation is mediated by endothelial dysfunction. Intrinsic vascular abnormalities may be important in sensitising HD patients to recurrent cumulative ischaemic end organ injury. PMID:25546407

  20. 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.

  1. Dissecting the Function and Assembly of Acentriolar Microtubule Organizing Centers in Drosophila Cells In Vivo

    PubMed Central

    Baumbach, Janina; Novak, Zsofia Anna; Raff, Jordan W.; Wainman, Alan

    2015-01-01

    Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2—the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs. PMID:26020779

  2. In vivo corneal confocal microscopic analysis in patients with keratoconus

    PubMed Central

    Bitirgen, Gulfidan; Ozkagnici, Ahmet; Bozkurt, Banu; Malik, Rayaz A

    2015-01-01

    AIM To quantify corneal ultrastructure using laser scanning in vivo confocal microscopy (IVCM) in patients with keratoconus and control subjects. METHODS Unscarred corneas of 78 keratoconic subjects without a history of contact lens use and 36 age-matched control subjects were evaluated with slit-lamp examination (SLE), corneal topography and laser scanning IVCM. One eye was randomly chosen for analysis. Anterior and posterior stromal keratocyte, endothelial cell and basal epithelial cell densities and sub-basal nerve structure were evaluated. RESULTS IVCM qualitatively demonstrated enlarged basal epithelial cells, structural changes in sub-basal and stromal nerve fibers, abnormal stromal keratocytes and keratocyte nuclei, and pleomorphism and enlargement of endothelial cells. Compared with control subjects, significant reductions in basal epithelial cell density (5817±306 cells/mm2 vs 4802±508 cells/mm2, P<0.001), anterior stromal keratocyte density (800±111 cells/mm2 vs 555±115 cells/mm2, P<0.001), posterior stromal keratocyte density (333±34 cells/mm2 vs 270±47 cells/mm2, P<0.001), endothelial cell density (2875±223 cells/mm2 vs 2686±265 cells/mm2, P<0.001), sub-basal nerve fiber density (31.2±8.4 nerves/mm2 vs 18.1±9.2 nerves/mm2, P<0.001), sub-basal nerve fiber length (21.4±3.4 mm/mm2 vs 16.1±5.1 mm/mm2, P<0.001), and sub-basal nerve branch density (median 50.0 (first quartile 31.2 - third quartile 68.7) nerve branches/mm2 vs median 25.0 (first quartile 6.2 - third quartile 45.3) nerve branches/mm2, P<0.001) were observed in patients with keratoconus. CONCLUSION Significant microstructural abnormalities were identified in all corneal layers in the eyes of subjects with keratoconus using IVCM. This non-invasive in vivo technique provides an important means to define and follow progress of microstructural changes in patients with keratoconus. PMID:26086003

  3. Analysis of topoisomerase function in bacterial replication fork movement: Use of

    E-print Network

    Botstein, David

    ( ) supercoils in vivo. Materials and Methods Bacterial Strains and Plasmids. The bacterial strains usedAnalysis of topoisomerase function in bacterial replication fork movement: Use of DNA microarrays of ( ) supercoils in plasmid DNA in vivo, suggesting that topo IV can promote replication by removing ( ) supercoils

  4. Analysis of the Ex Vivo and In Vivo Antiretroviral Activity of Gemcitabine

    PubMed Central

    Clouser, Christine L.; Holtz, Colleen M.; Mullett, Mary; Crankshaw, Duane L.; Briggs, Jacquie E.; Chauhan, Jay; VanHoutan, Ilze Matise; Patterson, Steven E.; Mansky, Louis M.

    2011-01-01

    Replication of retroviral and host genomes requires ribonucleotide reductase to convert rNTPs to dNTPs, which are then used as substrates for DNA synthesis. Inhibition of ribonucleotide reductase by hydroxyurea (HU) has been previously used to treat cancers as well as HIV. However, the use of HU as an antiretroviral is limited by its associated toxicities such as myelosuppression and hepatotoxicity. In this study, we examined the ribonucleotide reductase inhibitor, gemcitabine, both in cell culture and in C57Bl/6 mice infected with LP-BM5 murine leukemia virus (LP-BM5 MuLV, a murine AIDS model). Gemcitabine decreased infectivity of MuLV in cell culture with an EC50 in the low nanomolar range with no detectable cytotoxicity. Similarly, gemcitabine significantly decreased disease progression in mice infected with LP-BM5. Specifically, gemcitabine treatment decreased spleen size, plasma IgM, and provirus levels compared to LP-BM5 MuLV infected, untreated mice. Gemcitabine efficacy was observed at doses as low as 1 mg/kg/day in the absence of toxicity. Higher doses of gemcitabine (3 mg/kg/day and higher) were associated with toxicity as determined by a loss in body mass. In summary, our findings demonstrate that gemcitabine has antiretroviral activity ex vivo and in vivo in the LP-BM5 MuLV model. These observations together with a recent ex vivo study with HIV-1[1], suggest that gemcitabine has broad antiretroviral activity and could be particularly useful in vivo when used in combination drug therapy. PMID:21264291

  5. Functionalized gold nanoparticles: a detailed in vivo multimodal microscopic brain distribution study

    NASA Astrophysics Data System (ADS)

    Sousa, Fernanda; Mandal, Subhra; Garrovo, Chiara; Astolfo, Alberto; Bonifacio, Alois; Latawiec, Diane; Menk, Ralf Hendrik; Arfelli, Fulvia; Huewel, Sabine; Legname, Giuseppe; Galla, Hans-Joachim; Krol, Silke

    2010-12-01

    In the present study, the in vivo distribution of polyelectrolyte multilayer coated gold nanoparticles is shown, starting from the living animal down to cellular level. The coating was designed with functional moieties to serve as a potential nano drug for prion disease. With near infrared time-domain imaging we followed the biodistribution in mice up to 7 days after intravenous injection of the nanoparticles. The peak concentration in the head of mice was detected between 19 and 24 h. The precise particle distribution in the brain was studied ex vivo by X-ray microtomography, confocal laser and fluorescence microscopy. We found that the particles mainly accumulate in the hippocampus, thalamus, hypothalamus, and the cerebral cortex.In the present study, the in vivo distribution of polyelectrolyte multilayer coated gold nanoparticles is shown, starting from the living animal down to cellular level. The coating was designed with functional moieties to serve as a potential nano drug for prion disease. With near infrared time-domain imaging we followed the biodistribution in mice up to 7 days after intravenous injection of the nanoparticles. The peak concentration in the head of mice was detected between 19 and 24 h. The precise particle distribution in the brain was studied ex vivo by X-ray microtomography, confocal laser and fluorescence microscopy. We found that the particles mainly accumulate in the hippocampus, thalamus, hypothalamus, and the cerebral cortex. Electronic supplementary information (ESI) available: Fig. S1-S6. See DOI: 10.1039/c0nr00345j

  6. Compound Ex Vivo and In Silico Method for Hemodynamic Analysis of Stented Arteries

    E-print Network

    Daraio, Chiara

    Compound Ex Vivo and In Silico Method for Hemodynamic Analysis of Stented Arteries Farhad Rikhtegar the coronary arteries of ex vivo porcine hearts, performed vascular corrosion casting, acquired the vessel in detail the stent geometry, arterial tissue prolapse, radial and axial arterial deformation as well

  7. Analysis of telomerase catalytic subunit mutants in vivo and in vitro in Schizosaccharomyces pombe

    E-print Network

    Nakamura, Toru M.

    Analysis of telomerase catalytic subunit mutants in vivo and in vitro in Schizosaccharomyces pombe by Thomas R. Cech, April 25, 2000 The chromosome end-replicating enzyme telomerase is composed of a templateTE of telomerase-specific motif T had no phenotype in vivo or in vitro whereas mutation of a conserved amino acid

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

    PubMed Central

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

    2015-01-01

    Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large-scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co-evolution of the plasmid library and E. coli genome driving increased galactose utilization. Our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo. PMID:25762151

  9. Analysis of Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity.

    PubMed

    Moulin, Michael; Nguyen, Ginnie T D T; Scaife, Mark A; Smith, Alison G; Fitzpatrick, Teresa B

    2013-09-01

    Thiamin (vitamin B1) is an essential micronutrient needed as a cofactor for many central metabolic enzymes. Animals must have thiamin in their diet, whereas bacteria, fungi, and plants can biosynthesize it de novo from the condensation of a thiazole and a pyrimidine moiety. Although the routes to biosynthesize these two heterocycles are not conserved in different organisms, in all cases exogenous thiamin represses expression of one or more of the biosynthetic pathway genes. One important mechanism for this control is via thiamin-pyrophosphate (TPP) riboswitches, regions of the mRNA to which TPP can bind directly, thus facilitating fine-tuning to maintain homeostasis. However, there is little information on how modulation of riboswitches affects thiamin metabolism in vivo. Here we use the green alga, Chlamydomonas reinhardtii, which regulates both thiazole and pyrimidine biosynthesis with riboswitches in the THI4 (Thiamin 4) and THIC (Thiamin C) genes, respectively, to investigate this question. Our study reveals that regulation of thiamin metabolism is not the simple dogma of negative feedback control. Specifically, balancing the provision of both of the heterocycles of TPP appears to be an important requirement. Furthermore, we show that the Chlamydomonas THIC riboswitch is controlled by hydroxymethylpyrimidine pyrophosphate, as well as TPP, but with an identical alternative splicing mechanism. Similarly, the THI4 gene is responsive to thiazole. The study not only provides insight into the plasticity of the TPP riboswitches but also shows that their maintenance is likely to be a consequence of evolutionary need as a function of the organisms' environment and the particular pathway used. PMID:23959877

  10. 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

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

    PubMed Central

    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

    2015-01-01

    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. PMID:25715804

  12. Comparative Meta-Analysis of Transcriptomics Data during Cellular Senescence and In Vivo Tissue Ageing

    PubMed Central

    Voutetakis, Konstantinos; Gonos, Efstathios S.; Trougakos, Ioannis P.

    2015-01-01

    Several studies have employed DNA microarrays to identify gene expression signatures that mark human ageing; yet the features underlying this complicated phenomenon remain elusive. We thus conducted a bioinformatics meta-analysis on transcriptomics data from human cell- and biopsy-based microarrays experiments studying cellular senescence or in vivo tissue ageing, respectively. We report that coregulated genes in the postmitotic muscle and nervous tissues are classified into pathways involved in cancer, focal adhesion, actin cytoskeleton, MAPK signalling, and metabolism regulation. Genes that are differentially regulated during cellular senescence refer to pathways involved in neurodegeneration, focal adhesion, actin cytoskeleton, proteasome, cell cycle, DNA replication, and oxidative phosphorylation. Finally, we revealed genes and pathways (referring to cancer, Huntington's disease, MAPK signalling, focal adhesion, actin cytoskeleton, oxidative phosphorylation, and metabolic signalling) that are coregulated during cellular senescence and in vivo tissue ageing. The molecular commonalities between cellular senescence and tissue ageing are also highlighted by the fact that pathways that were overrepresented exclusively in the biopsy- or cell-based datasets are modules either of the same reference pathway (e.g., metabolism) or of closely interrelated pathways (e.g., thyroid cancer and melanoma). Our reported meta-analysis has revealed novel age-related genes, setting thus the basis for more detailed future functional studies. PMID:25977747

  13. Assembly requirements of PU.1-Pip (IRF-4) activator complexes: inhibiting function in vivo using fused dimers.

    PubMed Central

    Brass, A L; Zhu, A Q; Singh, H

    1999-01-01

    Gene expression in higher eukaryotes appears to be regulated by specific combinations of transcription factors binding to regulatory sequences. The Ets factor PU.1 and the IRF protein Pip (IRF-4) represent a pair of interacting transcription factors implicated in regulating B cell-specific gene expression. Pip is recruited to its binding site on DNA by phosphorylated PU.1. PU.1-Pip interaction is shown to be template directed and involves two distinct protein-protein interaction surfaces: (i) the ets and IRF DNA-binding domains; and (ii) the phosphorylated PEST region of PU.1 and a lysine-requiring putative alpha-helix in Pip. Thus, a coordinated set of protein-protein and protein-DNA contacts are essential for PU.1-Pip ternary complex assembly. To analyze the function of these factors in vivo, we engineered chimeric repressors containing the ets and IRF DNA-binding domains connected by a flexible POU domain linker. When stably expressed, the wild-type fused dimer strongly repressed the expression of a rearranged immunoglobulin lambda gene, thereby establishing the functional importance of PU.1-Pip complexes in B cell gene expression. Comparative analysis of the wild-type dimer with a series of mutant dimers distinguished a gene regulated by PU.1 and Pip from one regulated by PU.1 alone. This strategy should prove generally useful in analyzing the function of interacting transcription factors in vivo, and for identifying novel genes regulated by such complexes. PMID:10022840

  14. 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 ?v?3 integrins overexpressed in the angiogenic tumor vasculatures. Using in vivo NIR optical imaging of mice bearing pancreatic cancer xenografts, implanted both subcutaneously and orthotopically, we have demonstrated that systemically delivered cRGD-conjugated QDs, but not the unconjugated ones, can efficiently target and label the tumors with high signal-to-noise ratio. Histopathological analysis of major organs of the treated mice showed no evidence of systemic toxicity associated with these QDs. These experiments suggest that cRGD-conjugated NIR QDs can serve as safe and efficient probes for optical bioimaging of tumors in vivo. Furthermore, by co-encapsulating these QDs and anticancer drugs within these micelles, we have demonstrated a promising theranostic, nanosized platform for both cancer imaging and therapy.

  15. 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.

  16. Structural Determinants of Arabidopsis thaliana Hyponastic Leaves 1 Function In Vivo

    PubMed Central

    Burdisso, Paula; Milia, Fernando; Schapire, Arnaldo L.; Bologna, Nicolás G.; Palatnik, Javier F.; Rasia, Rodolfo M.

    2014-01-01

    MicroRNAs have turned out to be important regulators of gene expression. These molecules originate from longer transcripts that are processed by ribonuclease III (RNAse III) enzymes. Dicer proteins are essential RNAse III enzymes that are involved in the generation of microRNAs (miRNAs) and other small RNAs. The correct function of Dicer relies on the participation of accessory dsRNA binding proteins, the exact function of which is not well-understood so far. In plants, the double stranded RNA binding protein Hyponastic Leaves 1 (HYL1) helps Dicer Like protein (DCL1) to achieve an efficient and precise excision of the miRNAs from their primary precursors. Here we dissected the regions of HYL1 that are essential for its function in Arabidopsis thaliana plant model. We generated mutant forms of the protein that retain their structure but affect its RNA-binding properties. The mutant versions of HYL1 were studied both in vitro and in vivo, and we were able to identify essential aminoacids/residues for its activity. Remarkably, mutation and even ablation of one of the purportedly main RNA binding determinants does not give rise to any major disturbances in the function of the protein. We studied the function of the mutant forms in vivo, establishing a direct correlation between affinity for the pri-miRNA precursors and protein activity. PMID:25409478

  17. In Vivo Analysis of Engrafted Adult Hippocampal Neural Progenitors

    E-print Network

    cell behavior in vivo. Adult hippocampal neural progenitor cells (AHNPCs) are one potential source. Once thawed, aliquot can be stored at 4°C for up to 1 week. 4. Sterile cotton-plugged glass Pasteur pipettes. 5. 5-Bromo-2˘-deoxyuridine (BrdU, Sigma, St. Louis, MO). Prepare 5 mM stock solution in distilled

  18. 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 ...

  19. Dynamic in vivo analysis of drug induced actin cytoskeleton degradation by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Schnekenburger, Juergen; Bredebusch, Ilona; Langehanenberg, Patrik; Domschke, Wolfram; von Bally, Gert; Kemper, Björn

    2007-07-01

    The actin cytoskeleton mediates a variety of crucial cellular functions as migration, intracellular transport, exocytosis, endocytosis and force generation. The highly dynamic actin fibers are therefore targets for several drugs and toxins. However the study of actin interfering processes by standard microscopy techniques fails in the detailed resolution of dynamic spatial alterations required for a deeper understanding of toxic effects. Here we applied digital holographic microscopy in the online functional analysis of the actin cytoskeleton disrupting marine toxin Latrunculin B. SEM and fluorescence microscopy showed rapid Latrunculin B induced alterations in cell morphology and actin fiber degradation in pancreas tumor cells. The dynamic digital holographic in vivo analysis of the drug dependent cellular processes demonstrated differences in the actin cytoskeleton stability of highly differentiated and dedifferentiated pancreas tumor cell lines. The spatial resolution of the morphological alterations revealed unequal changes in cell morphology. While cells with a low metastatic potential showed Latrunculin B induced cell collapse within 4 h the metastatic tumor cells were increased in cell volume indicating Latrunculin B effects also on cell water content. These data demonstrate that marker free, non-destructive online analysis of cellular morphology and dynamic spatial processes in living cells by digital holography offers new insights in actin dependent cellular mechanisms. Digital holographic microscopy was shown to be a versatile tool in the screening of toxic drug effects and cancer cell biology.

  20. Functional data analysis in hydrology

    NASA Astrophysics Data System (ADS)

    Chebana, F.; Dabo-Niang, S.; Ouarda, T.

    2013-12-01

    River flow records are essential for the prevention of flood risks and the effective planning and management of water resources among other engineering activities. The graphical representation of the temporal variation of flow over a period of time constitutes a hydrograph. The latter is usually characterized by its peak, volume and duration. These features are considered jointly in order to take into account their dependence structure within multivariate hydrological frequency analysis (HFA). However, all these multivariate HFA approaches are based on the analysis of a limited number of characteristics and do not make use of the full information provided by the hydrograph. This talk is to propose to introduce a new framework for HFA using the hydrographs as curves to be functional data. In the context, called functional data analysis (FDA), the whole hydrograph is considered as one infinite-dimensional observation. The FDA context in HFA has a number of advantages. A number of functional tools are introduced and adapted to flood HFA with a focus on exploratory analysis. A real-world flood analysis case-study is considered.

  1. 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.

  2. The Intramembrane Proteases Signal Peptide Peptidase-Like 2a and 2b Have Distinct Functions In Vivo

    PubMed Central

    Schneppenheim, Janna; Hüttl, Susann; Mentrup, Torben; Lüllmann-Rauch, Renate; Rothaug, Michelle; Engelke, Michael; Dittmann, Kai; Dressel, Ralf; Araki, Masatake; Araki, Kimi; Wienands, Jürgen; Fluhrer, Regina; Saftig, Paul

    2014-01-01

    We reported recently that the presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is essential for B cell development by cleaving the N-terminal fragment (NTF) of the invariant chain (li, CD74). Based on this, we suggested that pharmacological modulation of SPPL2a may represent a novel approach to deplete B cells in autoimmune disorders. With regard to reported overlapping substrate spectra of SPPL2a and its close homologue, SPPL2b, we investigated the role of SPPL2b in CD74 NTF proteolysis and its impact on B and dendritic cell homeostasis. In heterologous expression experiments, SPPL2b was found to cleave CD74 NTF with an efficiency simliar to that of SPPL2a. For in vivo analysis, SPPL2b single-deficient and SPPL2a/SPPL2b double-deficient mice were generated and examined for CD74 NTF turnover/accumulation, B cell maturation and functionality, and dendritic cell homeostasis. We demonstrate that in vivo SPPL2b does not exhibit a physiologically relevant contribution to CD74 proteolysis in B and dendritic cells. Furthermore, we reveal that both proteases exhibit divergent subcellular localizations in B cells and different expression profiles in murine tissues. These findings suggest distinct functions of SPPL2a and SPPL2b and, based on a high abundance of SPPL2b in brain, a physiological role of this protease in the central nervous system. PMID:24492962

  3. S100A1 gene therapy preserves in vivo cardiac function after myocardial infarction.

    PubMed

    Pleger, Sven T; Remppis, Andrew; Heidt, Beatrix; Völkers, Mirko; Chuprun, J Kurt; Kuhn, Matthew; Zhou, Rui-Hai; Gao, Erhe; Szabo, Gabor; Weichenhan, Dieter; Müller, Oliver J; Eckhart, Andrea D; Katus, Hugo A; Koch, Walter J; Most, Patrick

    2005-12-01

    Myocardial infarction (MI) represents an enormous clinical challenge as loss of myocardium due to ischemic injury is associated with compromised left ventricular (LV) function often leading to acute cardiac decompensation or chronic heart failure. S100A1 was recently identified as a positive inotropic regulator of myocardial contractility in vitro and in vivo. Here, we explore the strategy of myocardial S100A1 gene therapy either at the time of, or 2 h after, MI to preserve global heart function. Rats underwent cryothermia-induced MI and in vivo intracoronary delivery of adenoviral transgenes (4 x 10(10) pfu). Animals received saline (MI), the S100A1 adenovirus (MI/AdS100A1), a control adenovirus (MI/AdGFP), or a sham operation. S100A1 gene delivery preserved global in vivo LV function 1 week after MI. Preservation of LV function was due mainly to S100A1-mediated gain of contractility of the remaining, viable myocardium since contractile parameters and Ca(2+) transients of isolated MI/AdS100A1 myocytes were significantly enhanced compared to myocytes isolated from both MI/AdGFP and sham groups. Moreover, S100A1 gene therapy preserved the cardiac beta-adrenergic inotropic reserve, which was associated with the attenuation of GRK2 up-regulation. Also, S100A1 overexpression reduced cardiac hypertrophy 1 week post-MI. Overall, our data indicate that S100A1 gene therapy provides a potential novel treatment strategy to maintain contractile performance of the post-MI heart. PMID:16168714

  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. Alterations at the Cross-Bridge Level Are Associated with a Paradoxical Gain of Muscle Function In Vivo in a Mouse Model of Nemaline Myopathy

    PubMed Central

    Gineste, Charlotte; Ottenheijm, Coen; Le Fur, Yann; Banzet, Sébastien; Pecchi, Emilie; Vilmen, Christophe; Cozzone, Patrick J.; Koulmann, Nathalie; Hardeman, Edna C.; Bendahan, David; Gondin, Julien

    2014-01-01

    Nemaline myopathy is the most common disease entity among non-dystrophic skeletal muscle congenital diseases. The first disease causing mutation (Met9Arg) was identified in the gene encoding ?-tropomyosinslow gene (TPM3). Considering the conflicting findings of the previous studies on the transgenic (Tg) mice carrying the TPM3Met9Arg mutation, we investigated carefully the effect of the Met9Arg mutation in 8–9 month-old Tg(TPM3)Met9Arg mice on muscle function using a multiscale methodological approach including skinned muscle fibers analysis and in vivo investigations by magnetic resonance imaging and 31-phosphorus magnetic resonance spectroscopy. While in vitro maximal force production was reduced in Tg(TPM3)Met9Arg mice as compared to controls, in vivo measurements revealed an improved mechanical performance in the transgenic mice as compared to the former. The reduced in vitro muscle force might be related to alterations occuring at the cross-bridges level with muscle-specific underlying mechanisms. In vivo muscle improvement was not associated with any changes in either muscle volume or energy metabolism. Our findings indicate that TPM3(Met9Arg) mutation leads to a mild muscle weakness in vitro related to an alteration at the cross-bridges level and a paradoxical gain of muscle function in vivo. These results clearly point out that in vitro alterations are muscle-dependent and do not necessarily translate into similar changes in vivo. PMID:25268244

  6. 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

  7. Comprehensive Analysis of in Vivo Phosphoproteome of Mouse Liver Microsomes.

    PubMed

    Kwon, Oh Kwang; Sim, JuHee; Kim, Sun Ju; Sung, Eunji; Kim, Jin Young; Jeong, Tae Cheon; Lee, Sangkyu

    2015-12-01

    Protein phosphorylation at serine, threonine, and tyrosine residues are some of the most widespread reversible post-translational modifications. Microsomes are vesicle-like bodies, not ordinarily present within living cells, which form from pieces of the endoplasmic reticulum (ER), plasma membrane, mitochondria, or Golgi apparatus of broken eukaryotic cells. Here we investigated the total phosphoproteome of mouse liver microsomes (MLMs) using TiO2 enrichment of phosphopeptides coupled to on-line 2D-LC-MS/MS. In total, 699 phosphorylation sites in 527 proteins were identified in MLMs. When compared with the current phosphoSitePlus database, 155 novel phosphoproteins were identified in MLM. The distributions of phosphosites were 89.4, 8.0, and 2.6% for phosphoserine, phosphotheronine, and phosphotyrosine, respectively. By Motif-X analysis, eight Ser motifs and one Thr motif were found, and five acidic, two basophilic-, and two proline-directed motifs were assigned. The potential functions of phosphoproteins in MLM were assigned by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In GO annotation, phosphorylated microsomal proteins were involved in mRNA processing, mRNA metabolic processes, and RNA splicing. In the KEGG pathway analysis, phosphorylated microsomal proteins were highly enriched in ribosome protein processing in ER and ribosomes and in RNA transport. Furthermore, we determined that 52 and 23 phosphoproteins were potential substrates of cAMP-dependent protein kinase A and casein kinase II, respectively, many of which are 40S/60S ribosomal proteins. Overall, our results provide an overview of features of protein phosphorylation in MLMs that should be a valuable resource for the future understanding of protein synthesis or translation involving phosphorylation. PMID:26487105

  8. 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. PMID:23684840

  9. 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

  10. 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.

  11. The Ras/Rap GTPase activating protein RASA3: from gene structure to in vivo functions.

    PubMed

    Schurmans, Stéphane; Polizzi, Séléna; Scoumanne, Ariane; Sayyed, Sufyan; Molina-Ortiz, Patricia

    2015-01-01

    RASA3 (or GTPase Activating Protein III, R-Ras GTPase-activating protein, GAP1(IP4BP)) is a GTPase activating protein of the GAP1 subfamily which targets Ras and Rap1. RASA3 was originally purified from pig platelet membranes through its intrinsic ability to bind inositol 1,3,4,5-tetrakisphosphate (I(1,3,4,5)P4) with high affinity, hence its first name GAP1(IP4BP) (for GAP1 subfamily member which binds I(1,3,4,5)P4). RASA3 was thus the first I(1,3,4,5)P4 receptor identified and cloned. The in vitro and in vivo functions of RASA3 remained somewhat elusive for a long time. However, recently, using genetically-modified mice and cells derived from these mice, the function of RASA3 during megakaryopoiesis, megakaryocyte adhesion and migration as well as integrin signaling has been reported. The goal of this review is thus to summarize and comment recent and less recent data in the literature on RASA3, in particular on the in vivo function of this specific GAP1 subfamily member. PMID:25294679

  12. Effects of antioxidants on endothelial function in human saphenous vein in an ex vivo model.

    PubMed

    Sharif, Muhammed Anees; Bayraktutan, Ulvi; Arya, Nityanand; Badger, Stephen A; O'Donnell, Mark E; Young, Ian S; Soong, Chee V

    2009-01-01

    This ex vivo study is aimed at determining the beneficial effects of antioxidant agents on human saphenous vein endothelial function. Vein rings harvested during infrainguinal bypass surgery were assessed in an organ bath for endothelium-dependent relaxation, initially without and then with the addition of 10 microM manganese tetrakis benzoic acid porphyrin (MnTBAP), 0.01% N-acetylcysteine (NAC), 0.02% NAC, 10 microM vitamin C, and 100 microM vitamin C. Fifty-five vein rings from 22 patients were analyzed. MnTBAP improved the endothelium-dependent relaxation when compared with control (57.0% vs 37.8%, P < .01). Addition of 0.01% or 0.02% NAC did not improve the endothelium-dependent vasorelaxation (28.2% vs 18.6%, P = ns and 37.8% vs 29.8%, P = ns, respectively). Although 10-microM vitamin C failed to improve endothelial function (50.6% vs 37.2%, P = ns), 100-microM vitamin C significantly enhanced endothelium-dependent relaxation (66.5% vs 38.3%, P < .001). These results suggest that the addition of MnTBAP and high-dose vitamin C can improve the endothelial function of harvested saphenous vein segments in an ex vivo model. PMID:18796454

  13. Photoacoustics and fluorescence based nanoprobes towards functional and structural imaging in vivo

    NASA Astrophysics Data System (ADS)

    Ray, Aniruddha

    Imaging of chemical analytes and structural properties related to physiological activities within biological systems is of great bio-medical interest; it can contribute to the fundamental understanding of biological systems and can be applied to the diagnosis and prognosis of diseases, especially tumors. The work presented in this thesis focuses on the development and application of polymeric nanoprobe aided optical imaging of chemical analytes (Oxygen, pH) and structural properties in live cells and animal models. To this end, specific nanoprobes, based on the polyacrylamide nanoplatform, bearing both appropriate targeting functionalities, and high concentrations of sensing and contrast agents, have been developed. The nanoprobes presented here are biodegradable, biocompatible and non-toxic, rendering them safe for in vivo use. Furthermore the nanoprobes are designed to have variable optical properties that are dependent on the local concentration of the specific analyte of interest. Optical imaging techniques that are particularly suited for deep tissue applications, such as two-photon fluorescence and photoacoustics, were applied for non-invasive real-time imaging and sensing in cancer cells, tumor spheroids and animal models. Our results demonstrate that this technique enables high sensitive detection of chemical analytes with a sensitivity of <5 Torr for oxygen and <0.1 pH units in vivo, which is better than the currently available in vivo functional imaging techniques. This non-invasive and non-ionizing, yet low cost, method will enable morphological and functional evaluation across any tissue, with both high spatial and temporal resolution but without eliciting short- or long-term tissue damage. Currently no gold standard exists for such xii functional imaging. The approach presented here can be used for early detection and diagnosis of tumors, as well as for monitoring the progression of disease and therapy. This technique will also enable observing phenomena at the cellular level in vivo that would lead to a better understanding of the pathophysiology of diseases as well as the disease onset, progression, and response to therapy.

  14. 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

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

    PubMed

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

    2015-11-01

    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

  16. In vivo functions of Drp1: Lessons learned from yeast genetics and mouse knockouts

    PubMed Central

    Sesaki, Hiromi; Adachi, Yoshihiro; Kageyama, Yusuke; Itoh, Kie; Iijima, Miho

    2014-01-01

    Mitochondria grow, divide, and fuse in cells. Mitochondrial division is critical for the maintenance of the structure and function of mitochondria. Alterations in this process have been linked to many human diseases, including peripheral neuropathies and aging-related neurological disorders. In this review, we discuss recent progress in mitochondrial division by focusing on molecular and in vivo analyses of the evolutionarily conserved, central component of mitochondrial division, dynamin-related protein 1 (Drp1), in the yeast and mouse model organisms. PMID:24326103

  17. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    NASA Astrophysics Data System (ADS)

    Vogel, R. F.; Linke, K.; Teichert, H.; Ehrmann, M. A.

    2008-07-01

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property.

  18. GPU Accelerated Statistical Analysis of Cryptographic Functions

    E-print Network

    Kaminsky, Alan

    Kaminsky (RIT) Statistical Analysis of Crypto Functions SIAM PP12 1 / 32 #12;Outline 1 Cryptographic Hash Alan Kaminsky (RIT) Statistical Analysis of Crypto Functions SIAM PP12 2 / 32 #12;Cryptographic Hash function's security is questionable Alan Kaminsky (RIT) Statistical Analysis of Crypto Functions SIAM PP12

  19. 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…

  20. 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?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. PMID:26467242

  1. Histone acetyltransferase activity and interaction with ADA2 are critical for GCN5 function in vivo.

    PubMed Central

    Candau, R; Zhou, J X; Allis, C D; Berger, S L

    1997-01-01

    Yeast GCN5 is one component of a putative adaptor complex that includes ADA2 and ADA3 and functionally connects DNA-bound transcriptional activators with general transcription factors. GCN5 possesses histone acetyltransferase (HAT) activity, conceptually linking transcriptional activation with enzymatic modification at chromatin. We have identified the minimal catalytic domain within GCN5 necessary to confer HAT activity and have shown that in vivo activity of GCN5 requires this domain. However, complementation of growth and transcriptional activation in gcn5- cells required not only the HAT domain of GCN5, but also interaction with ADA2. The bromodomain in GCN5 was dispensable for HAT activity and for transcriptional activation by strong activators; however, it was required for full complementation in other assays. Fusion of GCN5 to the bacterial lexA DNA binding domain activated transcription in vivo, and required both the HAT domain and the ADA2 interaction domain. These results suggest that both functions of GCN5, HAT activity and interaction with ADA2, are necessary for targeting and acetylation of nucleosomal histones. PMID:9034338

  2. Ubiquitination regulates the neuroprotective function of the deubiquitinase ataxin-3 in vivo.

    PubMed

    Tsou, Wei-Ling; Burr, Aaron A; Ouyang, Michelle; Blount, Jessica R; Scaglione, K Matthew; Todi, Sokol V

    2013-11-29

    Deubiquitinases (DUBs) are proteases that regulate various cellular processes by controlling protein ubiquitination. Cell-based studies indicate that the regulation of the activity of DUBs is important for homeostasis and is achieved by multiple mechanisms, including through their own ubiquitination. However, the physiological significance of the ubiquitination of DUBs to their functions in vivo is unclear. Here, we report that ubiquitination of the DUB ataxin-3 at lysine residue 117, which markedly enhances its protease activity in vitro, is critical for its ability to suppress toxic protein-dependent degeneration in Drosophila melanogaster. Compared with ataxin-3 with only Lys-117 present, ataxin-3 that does not become ubiquitinated performs significantly less efficiently in suppressing or delaying the onset of toxic protein-dependent degeneration in flies. According to further studies, the C terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase that ubiquitinates ataxin-3 in vitro, is dispensable for its ubiquitination in vivo and is not required for the neuroprotective function of this DUB in Drosophila. Our work also suggests that ataxin-3 suppresses degeneration by regulating toxic protein aggregation rather than stability. PMID:24106274

  3. 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.

  4. Thrombin as a multi-functional enzyme. Focus on in vitro and in vivo effects.

    PubMed

    Siller-Matula, Jolanta M; Schwameis, Michael; Blann, Andrew; Mannhalter, Christine; Jilma, Bernd

    2011-12-01

    Thrombin is the central protease in the coagulation cascade and one of the most extensively studied of all enzymes. In addition to its recognised role in the coagulation cascade and haemostasis, thrombin is known to have multiple pleiotropic effects, which mostly have been shown only in in vitro studies: it plays a role in inflammation and cellular proliferation and displays a mitogen activity on smooth muscle cells and endothelial cells, predominantly by activation of angiogenesis. In vivo , thrombin effects were examined in animal models of intravenous or intraarterial thrombin infusion. An extensive literature search regarding in vivo data showed that i) thrombin administered as a bolus causes microembolism, ii) thrombin infused slowly at steady-state conditions (up to 1.6 U/kg/min) leads to bleeds but not to intravascular clotting, iii) large quantity of thrombin infused at low rates (0.05 U/kg/min) does not have any measurable effect, and iv) thrombin increases vascular permeability leading to tissue damage. Although several decades of research on thrombin functions have provided a framework for understanding the biology of thrombin, animal and human studies with use of newer laboratory techniques are still needed to confirm the pleiotropic thrombin functions shown in in vitro studies. PMID:21979864

  5. In Vivo analysis of 14-3-3 proteins 

    E-print Network

    Philip, Nisha

    2000-01-01

    The 14-3-3 proteins are small acidic cytosolic proteins abundant in the nervous system, but without a well established physiological function. Putative functions assigned to these proteins include regulation of Ca˛? exocytosis, and activity...

  6. 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.

  7. 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.

  8. 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-?) 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-? 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 partly to its protective effects for the immune function. PMID:24090456

  9. The yeast centromere CDEI/Cpf1 complex: differences between in vitro binding and in vivo function.

    PubMed Central

    Wilmen, A; Pick, H; Niedenthal, R K; Sen-Gupta, M; Hegemann, J H

    1994-01-01

    The centromere and promoter factor Cpf1 binds centromere DNA element I found in all centromere DNAs from the yeast Saccharomyces cerevisiae. We analyzed thirty different point mutations in or around CEN6-CDEI (ATCACGTG) for their relative binding affinity to Cpf1 and these data were compared with the in vivo centromere function of these mutants. We show that the minimal length of the Cpf1 binding site needed for full in vitro binding and in vivo activity is 10 base pairs long comprised of CDEI plus the two base pairs 3' of this sequence. The palindromic core sequence CACGTG is most important for in vivo CEN function and in vitro Cpf1 binding. Symmetrical mutations in either halfsite of the core sequence affect in vitro Cpf1 binding and in vivo mitotic centromere function asymmetrically albeit to a different extent. Enlarging the CDEI palindrome to 12 or 20 bps increases in vitro Cpf1 binding but results in increased chromosome loss rates suggesting a need for asymmetrical Cpf1 binding sequences. Additionally, the ability of Cpf1 protein to bind a mutant CDEI element in vitro does not parallel the ability of that mutant to confer in vivo CEN activity. Our data indicate that the in vitro binding characteristics of Cpf1 to CDEI only partly overlap with their corresponding activity within the centromere complex, thus suggesting that in the in vivo situation the CDEI/Cpf1 complex might undergo interactions with other centromere DNA/protein complexes. Images PMID:8052535

  10. 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 ?g/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 demonstrates comparable in vivo sequestration efficacy at low exposure concentrations. The comparable response may be the result of the low 137Cs chloride dose and high sorbent dosage that was utilized. Future studies are planned to optimize SAMMS in vivo performance over a broader range of doses and conditions. PMID:20699707

  11. 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 ?geq/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 ?geq/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 low exposure concentrations. The comparable response may be the result of the low 137Cs dose and high sorbent dosage that was utilized. Future studies are planned to optimize SAMMS in vivo performance over a broader range of doses and conditions.

  12. Models in palaeontological functional analysis

    PubMed Central

    Anderson, Philip S. L.; Bright, Jen A.; Gill, Pamela G.; Palmer, Colin; Rayfield, Emily J.

    2012-01-01

    Models are a principal tool of modern science. By definition, and in practice, models are not literal representations of reality but provide simplifications or substitutes of the events, scenarios or behaviours that are being studied or predicted. All models make assumptions, and palaeontological models in particular require additional assumptions to study unobservable events in deep time. In the case of functional analysis, the degree of missing data associated with reconstructing musculoskeletal anatomy and neuronal control in extinct organisms has, in the eyes of some scientists, rendered detailed functional analysis of fossils intractable. Such a prognosis may indeed be realized if palaeontologists attempt to recreate elaborate biomechanical models based on missing data and loosely justified assumptions. Yet multiple enabling methodologies and techniques now exist: tools for bracketing boundaries of reality; more rigorous consideration of soft tissues and missing data and methods drawing on physical principles that all organisms must adhere to. As with many aspects of science, the utility of such biomechanical models depends on the questions they seek to address, and the accuracy and validity of the models themselves. PMID:21865242

  13. 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

  14. Functional Evaluation of ES–Somatic Cell Hybrids In Vitro and In Vivo

    PubMed Central

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

    2014-01-01

    Abstract 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. PMID:24787484

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

    PubMed

    Donovan, Kirk W; Bretscher, Anthony

    2015-05-11

    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

  16. 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.

  17. In Vitro Hematological and In Vivo Vasoactivity Assessment of Dextran Functionalized Graphene

    PubMed Central

    Chowdhury, Sayan Mullick; Kanakia, Shruti; Toussaint, Jimmy D.; Frame, Mary D.; Dewar, Anthony M.; Shroyer, Kenneth R.; Moore, William; Sitharaman, Balaji

    2013-01-01

    The intravenous, intramuscular or intraperitoneal administration of water solubilized graphene nanoparticles for biomedical applications will result in their interaction with the hematological components and vasculature. Herein, we have investigated the effects of dextran functionalized graphene nanoplatelets (GNP-Dex) on histamine release, platelet activation, immune activation, blood cell hemolysis in vitro, and vasoactivity in vivo. The results indicate that GNP-Dex formulations prevented histamine release from activated RBL-2H3 rat mast cells, and at concentrations ? 7?mg/ml, showed a 12–20% increase in levels of complement proteins. Cytokine (TNF-Alpha and IL-10) levels remained within normal range. GNP-Dex formulations did not cause platelet activation or blood cell hemolysis. Using the hamster cheek pouch in vivo model, the initial vasoactivity of GNP-Dex at concentrations (1–50?mg/ml) equivalent to the first pass of a bolus injection was a brief concentration-dependent dilation in arcade and terminal arterioles. However, they did not induce a pro-inflammatory endothelial dysfunction effect. PMID:24002570

  18. Feasibility of measuring selenium in humans using in vivo neutron activation analysis.

    PubMed

    Tahir, S N A; Chettle, D R; Byun, S H; Prestwich, W V

    2015-11-01

    Selenium (Se) is an element that, in trace quantities, plays an important role in the normal function of a number of biological processes in humans. Many studies have demonstrated that selenium deficiency in the body may contribute to an increased risk for certain neoplastic, cardiovascular, osseous, and nervous system diseases including retardation of bone formation. However, at higher concentrations Se is cytotoxic. For these reasons it is desirable to have a means of monitoring selenium concentration in humans.This paper presents the outcome of a feasibility study carried out for measuring selenium in humans using in vivo neutron activation analysis (IVNAA). In this technique a small dose of neutrons is delivered to the organ of interest, the neutrons are readily captured by the target nuclei, and the ?-rays given off are detected outside of the body. For the present study, human hand (bone) tissue equivalent phantoms were prepared with varying amounts of Se. These were irradiated by a low energy fast neutron beam produced by the (7)Li(p,n)(7)Be reaction employing the high beam current Tandetron accelerator. The counting data saved using a 4? NaI(TI) detection system were analyzed. The selenium was detected via the neutron capture reaction, (76)Se(n,?)(77m)Se, whereas calcium was detected through the (48)Ca(n,?)(49)Ca reaction for the purpose of normalization of the Se signals to the calcium signals. From the calibration lines drawn between Se/Ca concentrations and Se/Ca counts ratio, the minimum detection limits (MDLs) were computed for two sets of phantoms irradiated under different irradiation parameters.In this study the optimized MDL value was determined to be 81?ng g(-1) (Se/phantom mass) for an equivalent dose of 188 mSv to the phantom. This MDL was found at least 10 times lower than the reported data on Se concentration measured in bone tissues. It was concluded that the NAA technique would be a feasible means of performing in vivo measurements of selenium in humans. Currently the data on in vivo measurement of selenium in humans are limited; the results of the present study would greatly contribute to the present data. PMID:26393663

  19. c-Kit Receptor Signaling Regulates Islet Vasculature, ?-Cell Survival, and Function In Vivo.

    PubMed

    Feng, Zhi-Chao; Popell, Alex; Li, Jinming; Silverstein, Jenna; Oakie, Amanda; Yee, Siu-Pok; Wang, Rennian

    2015-11-01

    The receptor tyrosine kinase c-Kit plays an integral role in maintaining ?-cell mass and function. Although c-Kit receptor signaling promotes angiogenesis in multiple cell types, its role in islet vasculature is unknown. This study examines the effects of c-Kit-mediated vascular endothelial growth factor isoform A (VEGF-A) and islet vascularization on ?-cell function and survival using in vitro cell culture and in vivo mouse models. In cultured INS-1 cells and primary islets, c-Kit regulates VEGF-A expression via the Akt/mammalian target of rapamycin (mTOR) signaling pathway. Juvenile mice with mutated c-Kit (c-Kit(Wv/+)) showed impaired islet vasculature and ?-cell dysfunction, while restoring c-Kit expression in ?-cells of c-Kit(Wv/+) mice rescued islet vascular defects through modulation of the Akt/mTOR/VEGF-A pathway, indicating that c-Kit signaling in ?-cells is a required regulator for maintaining normal islet vasculature. Furthermore, ?-cell-specific c-Kit overexpression (c-Kit?Tg) in aged mice showed significantly increased islet vasculature and ?-cell function, but, when exposed to a long-term high-fat diet, c-Kit signaling in c-Kit?Tg mice induced substantial vascular remodeling, which resulted in increased islet inflammatory responses and ?-cell apoptosis. These results suggest that c-Kit-mediated VEGF-A action in ?-cells plays a pivotal role in maintaining islet vascularization and function. PMID:26253609

  20. Analysis of elastography methods using mathematical and ex vivo data

    NASA Astrophysics Data System (ADS)

    Byram, Brett C.; Wahl, Michael R.; Holmes, David R., III; Lerman, Amir; Robb, Richard A.

    2003-05-01

    Intravascular ultrasound (IVUS) currently has a limited ability to characterize endovascular anatomic properties. IVUS elastography enhances the ability to characterize the biomechanical properties of arterial walls. A mathematical phantom generator was developed based on the characteristics of 30MHz, 64 element IVUS catheter images from excised canine femoral arteries. The difference between high and low-pressure intra-arterial images was modeled using phase shifts. The increase in phase shift occurred randomly, generally at every three pixels in our images. Using mathematical phantoms, different methods for calculating elastograms were quantitatively analyzed. Specifically, the effect of standard cross correlation versus cross correlation of the integral of the inflection characteristics for a given set of data, and the effect of an algorithm utilizing a non-constant kernel, were assessed. The specific methods found to be most accurate on the mathematical phantom data were then applied to ex vivo canine data of a scarred and a healthy artery. The algorithm detected significant differences between these two sets of arterial data. It will be necessary to obtain and analyze several more sets of canine arterial data in order to determine the accuracy and reproducibility of the algorithm.

  1. Improving the signal analysis for in vivo photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Niu, Zhenyu; Yang, Ping; Wei, Dan; Tang, Shuo; Wei, Xunbin

    2015-03-01

    At early stage of cancer, a small number of circulating tumor cells (CTCs) appear in the blood circulation. Thus, early detection of malignant circulating tumor cells has great significance for timely treatment to reduce the cancer death rate. We have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of CTCs and record the signals from target cells. Information of target cells which is helpful to the early therapy would be obtained through analyzing and processing the signals. The raw signal detected from target cells often contains some noise caused by electronic devices, such as background noise and thermal noise. We choose the Wavelet denoising method to effectively distinguish the target signal from background noise. Processing in time domain and frequency domain would be combined to analyze the signal after denoising. This algorithm contains time domain filter and frequency transformation. The frequency spectrum image of the signal contains distinctive features that can be used to analyze the property of target cells or particles. The PAFC technique can detect signals from circulating tumor cells or other particles. The processing methods have a great potential for analyzing signals accurately and rapidly.

  2. 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 ?m (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.

  3. Influence of endothelium-derived relaxing factor on platelet function and hemostasis in vivo.

    PubMed

    Houston, D S; Buchanan, M R

    1994-04-01

    Endothelium-derived relaxing factor (EDRF) is a potent vasodilator, and is also, in vitro, a platelet-inhibitor. Experiments were performed to determine whether systemically released EDRF inhibits platelet-dependent hemostasis in vivo. Rabbits were treated with agents to release or block EDRF, and 5 standardized incisions were made in the ear. Carbachol, infused to stimulate EDRF release, abruptly lowered the blood pressure and caused increased bleeding. Neither effect was attributable to prostacyclin since neither was blocked by treatment of the rabbits with acetylsalicylic acid. In contrast, both the hypotension and bleeding were attenuated by the selective antagonist of EDRF synthesis, NG-nitro-L-arginine. However, neither the hypotension nor the bleeding associated with carbachol was inhibited by an infusion of free hemoglobin, used to scavenge intraluminally-released EDRF. We conclude that in this model endogenously-released EDRF increases bleeding indirectly by provoking vasodilatation, rather than directly by inhibiting platelet function. PMID:8029806

  4. 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-PPAR? ligand binding domain (LBD) fusion protein-luciferase reporter assay to examine PPAR? 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 PPAR? LBD and enhanced lipid accumulation in differentiating 3T3-L1 cells. SCO increased the transcription of several PPAR? target genes in differentiating 3T3-L1 cells and rescued the negative effects of tumor necrosis factor ? 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 AMPK? 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 effects on adipocytes in vitro and adipose tissue in vivo, highlighting its potential as a metabolically favorable botanical supplement. PMID:24915004

  5. In vivo analysis of intestinal permeability following hemorrhagic shock

    PubMed Central

    Alsaigh, Tom; Chang, Marisol; Richter, Michael; Mazor, Rafi; Kistler, Erik B

    2015-01-01

    AIM: To determine the time course of intestinal permeability changes to proteolytically-derived bowel peptides in experimental hemorrhagic shock. METHODS: We injected fluorescently-conjugated casein protein into the small bowel of anesthetized Wistar rats prior to induction of experimental hemorrhagic shock. These molecules, which fluoresce when proteolytically cleaved, were used as markers for the ability of proteolytically cleaved intestinal products to access the central circulation. Blood was serially sampled to quantify the relative change in concentration of proteolytically-cleaved particles in the systemic circulation. To provide spatial resolution of their location, particles in the mesenteric microvasculature were imaged using in vivo intravital fluorescent microscopy. The experiments were then repeated using an alternate measurement technique, fluorescein isothiocyanate (FITC)-labeled dextrans 20, to semi-quantitatively verify the ability of bowel-derived low-molecular weight molecules (< 20 kD) to access the central circulation. RESULTS: Results demonstrate a significant increase in systemic permeability to gut-derived peptides within 20 min after induction of hemorrhage (1.11 ± 0.19 vs 0.86 ± 0.07, P < 0.05) compared to control animals. Reperfusion resulted in a second, sustained increase in systemic permeability to gut-derived peptides in hemorrhaged animals compared to controls (1.2 ± 0.18 vs 0.97 ± 0.1, P < 0.05). Intravital microscopy of the mesentery also showed marked accumulation of fluorescent particles in the microcirculation of hemorrhaged animals compared to controls. These results were replicated using FITC dextrans 20 [10.85 ± 6.52 vs 3.38 ± 1.11 fluorescent intensity units (× 105, P < 0.05, hemorrhagic shock vs controls)], confirming that small bowel ischemia in response to experimental hemorrhagic shock results in marked and early increases in gut membrane permeability. CONCLUSION: Increased small bowel permeability in hemorrhagic shock may allow for systemic absorption of otherwise retained proteolytically-generated peptides, with consequent hemodynamic instability and remote organ failure. PMID:26557479

  6. 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

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

    PubMed

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

    2015-09-01

    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-sectional design. Ageing was found to be related to decreased leak regardless of training status. Increased training status was associated with increased mitochondrial hydrogen peroxide emission. 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. H2 O2 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. PMID:26096709

  8. Quantitative Analysis of Peristaltic and Segmental Motion In Vivo in the Rat Small Intestine Using Dynamic

    E-print Network

    Brasseur, James G.

    Quantitative Analysis of Peristaltic and Segmental Motion In Vivo in the Rat Small Intestine Using of nutrients that takes place within the small intestine. The normal processes of the small intestine are known been used extensively to study segments of the intestine that have been exteriorized from animals

  9. In Vivo Risk Analysis of Pancreatic Cancer Through Optical Characterization of Duodenal Mucosa

    E-print Network

    Hartline, Jason D.

    In Vivo Risk Analysis of Pancreatic Cancer Through Optical Characterization of Duodenal Mucosa,§ and Vadim Backman, PhD* Objectives: To reduce pancreatic cancer mortality, a paradigm shift in cancer) spectroscopy to predict the presence of pancreatic cancer by interrogating the duodenal mucosa. A previous ex

  10. In Vivo Bioavailability and In Vitro Bioaccessibility of Perfluorooctanoic Acid (PFOA) in Food Matrices: Correlation Analysis

    E-print Network

    Ma, Lena

    In Vivo Bioavailability and In Vitro Bioaccessibility of Perfluorooctanoic Acid (PFOA) in Food Matrices: Correlation Analysis and Method Development Kan Li, Chao Li, Nan-Yang Yu, Albert L. Juhasz, Xin ABSTRACT: Food is a major source of human exposure to perfluorooctanoic acid (PFOA), however, PFOA

  11. 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...

  12. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26612326

  14. 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.

  15. Structure predicts function: Combining non-invasive electrophysiology with in-vivo histology

    PubMed Central

    Helbling, Saskia; Teki, Sundeep; Callaghan, Martina F.; Sedley, William; Mohammadi, Siawoosh; Griffiths, Timothy D.; Weiskopf, Nikolaus; Barnes, Gareth R.

    2015-01-01

    We present an approach for combining high resolution MRI-based myelin mapping with functional information from electroencephalography (EEG) or magnetoencephalography (MEG). The main contribution to the primary currents detectable with EEG and MEG comes from ionic currents in the apical dendrites of cortical pyramidal cells, aligned perpendicularly to the local cortical surface. We provide evidence from an in-vivo experiment that the variation in MRI-based myeloarchitecture measures across the cortex predicts the variation of the current density over individuals and thus is of functional relevance. Equivalent current dipole locations and moments due to pitch onset evoked response fields (ERFs) were estimated by means of a variational Bayesian algorithm. The myeloarchitecture was estimated indirectly from individual high resolution quantitative multi-parameter maps (MPMs) acquired at 800 ?m isotropic resolution. Myelin estimates across cortical areas correlated positively with dipole magnitude. This correlation was spatially specific: regions of interest in the auditory cortex provided significantly better models than those covering whole hemispheres. Based on the MPM data we identified the auditory cortical area TE1.2 as the most likely origin of the pitch ERFs measured by MEG. We can now proceed to exploit the higher spatial resolution of quantitative MPMs to identify the cortical origin of M/EEG signals, inform M/EEG source reconstruction and explore structure–function relationships at a fine structural level in the living human brain. PMID:25529007

  16. 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

    2015-01-01

    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. PMID:26612326

  17. Monitoring of in vivo function of superparamagnetic iron oxide labelled murine dendritic cells during anti-tumour vaccination.

    PubMed

    Tavaré, Richard; Sagoo, Pervinder; Varama, Gopal; Tanriver, Yakup; Warely, Alice; Diebold, Sandra S; Southworth, Richard; Schaeffter, Tobias; Lechler, Robert I; Razavi, Reza; Lombardi, Giovanna; Mullen, Gregory E D

    2011-01-01

    Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4(+) T cells but, most importantly, they primed cytotoxic CD8(+) T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer. PMID:21637760

  18. Transcriptome Analysis in Chicken Cecal Epithelia upon Infection by Eimeria tenella In Vivo

    PubMed Central

    Guo, Aijiang; Cai, Jianping; Gong, Wei; Yan, Hongbin; Luo, Xuenong; Tian, Guangfu; Zhang, Shaohua; Zhang, Haili; Zhu, Guan; Cai, Xuepeng

    2013-01-01

    Coccidiosis, caused by various Eimeria species, is a major parasitic disease in chickens. However, our understanding on how chickens respond to coccidian infection is highly limited at both molecular and cellular levels. The present study employed the Affymetrix chicken genome array and performed transcriptome analysis on chicken cecal epithelia in response to infection for 4.5 days in vivo by the cecal-specific species E. tenella. By Significance Analysis of Microarrays (SAM), we have identified 7,099 probe sets with q-values at <0.05, in which 4,033 and 3,066 genes were found to be up- or down-regulated in response to parasite infection. The reliability of the microarray data were validated by real-time qRT-PCR of 20 genes with varied fold changes in expression (i.e., correlation coefficient between microarray and qRT-PCR datasets: R2?=?0.8773, p<0.0001). Gene ontology analysis, KEGG pathway mapping and manual annotations of regulated genes indicated that up-regulated genes were mainly involved in immunity/defense, responses to various stimuli, apoptosis/cell death and differentiation, signal transduction and extracellular matrix (ECM), whereas down-regulated genes were mainly encoding general metabolic enzymes, membrane components, and some transporters. Chickens mustered complex cecal eipthelia molecular and immunological responses in response to E. tenella infection, which included pathways involved in cytokine production and interactions, natural killer cell mediated cytotoxicity, and intestinal IgA production. In response to the pathogenesis and damage caused by infection, chicken cecal epithelia reduced general metabolism, DNA replication and repair, protein degradation, and mitochondrial functions. PMID:23737974

  19. 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…

  20. The past, present, and future of x-ray technology for in vivo imaging of function and form

    SciTech Connect

    Fouras, A.; Dubsky, S.; Hourigan, K.; Kitchen, M. J.; Lewis, R. A.; Hooper, S. B.

    2009-05-15

    Scientists and clinicians have a keen interest in studying not just the structure of physiological systems, but their motion also, or more generally their form and function. This paper focuses on the technologies that underpin in vivo measurements of form and function of the human body for both research and medical treatment. A concise literature review of x-ray imaging, ultrasonography, magnetic resonance imaging, radionuclide imaging, laser Doppler velocimetry, and particle image velocimetry is presented. Additionally, a more detailed review of in vivo x-ray imaging is presented. Finally, two techniques, which the authors believe are representative of the present and future of in vivo x-ray imaging techniques, are presented.

  1. In VivoFunctional Imaging of Intrinsic Scattering Changes in the Human Retina with High-speed Ultrahigh Resolution OCT

    PubMed Central

    Srinivasan, V. J.; Chen, Y.; Duker, J. S.; Fujimoto, J. G.

    2009-01-01

    Non-invasive methods of probing retinal function are of interest for the early detection of retinal disease. While retinal function is traditionally directly measured with the electroretinogram (ERG), recently functional optical imaging of the retina has been demonstrated. In this manuscript, stimulus-induced, intrinsic optical scattering changes in the human retina are measured in vivo with high-speed, ultrahigh resolution optical coherence tomography (OCT) operating at 50,000 axial scans per second and ?3.3 micron axial resolution. A stimulus and measurement protocol that enables measurement of functional OCT retinal signals is described. OCT signal changes in the photoreceptors are demonstrated. Two distinct responses having different temporal and spatial properties are reported. These results are discussed in the context of optical intrinsic signals measured previously in the retina by fundus imaging and scanning laser ophthalmoscopy. Finally, challenges associated with in vivo functional retinal imaging in human subjects are discussed. PMID:19259228

  2. In vitro, in vivo and ex vivo characterization of ibrutinib: a potent inhibitor of the efflux function of the transporter MRP1

    PubMed Central

    Zhang, Hui; Patel, Atish; Ma, Shao-Lin; Li, Xiao Jie; Zhang, Yun-Kai; Yang, Pei-Qi; Kathawala, Rishil J; Wang, Yi-Jun; Anreddy, Nagaraju; Fu, Li-Wu; Chen, Zhe-Sheng

    2014-01-01

    Background and Purpose The transporter, multidrug resistance protein 1 (MRP1, ABCC1), plays a critical role in the development of multidrug resistance (MDR). Ibrutinib is an inhibitor of Bruton's tyrosine kinase. Here we investigated the reversal effect of ibrutinib on MRP1-mediated MDR. Experimental Approach Cytotoxicity was determined by MTT assay. The expression of protein was detected by Western blot. RT-PCR and Q-PCR were performed to detect the expression of MRP1 mRNA. The intracellular accumulation and efflux of substrates for MRP1 were measured by scintillation counter and flow cytometry. HEK293/MRP1 cell xenografts in nude mice were established to study the effects of ibrutinib in vivo. Key Results Ibrutinib significantly enhanced the cytotoxicity of MRP1 substrates in HEK293/MRP1 and HL60/Adr cells overexpressing MRP1. Furthermore, ibrutinib increased the accumulation of substrates in these MRP1-overexpressing cells by inhibiting the drug efflux function of MRP1. However, mRNA and protein expression of MRP1 remained unaltered after treatment with ibrutinib in MRP1-overexpressing cells. In vivo, ibrutinib enhanced the efficacy of vincristine to inhibit the growth of HEK293/MRP1 tumour xenografts in nude mice. Importantly, ibrutinib also enhances the cytotoxicity of vincristine in primary cultures of leukaemia blasts, derived from patients. Conclusions and Implications Our results indicated that ibrutinib significantly increased the efficacy of the chemotherapeutic agents which were MRP1 substrates, in MRP1-overexpressing cells, in vitro, in vivo and ex vivo. These findings will lead to further studies on the effects of a combination of ibrutinib with chemotherapeutic agents in cancer patients overexpressing MRP1. PMID:25164592

  3. Stomatin-Like Protein 2 Is Required for In Vivo Mitochondrial Respiratory Chain Supercomplex Formation and Optimal Cell Function

    PubMed Central

    Mitsopoulos, Panagiotis; Chang, Yu-Han; Wai, Timothy; König, Tim; Dunn, Stanley D.; Langer, Thomas

    2015-01-01

    Stomatin-like protein 2 (SLP-2) is a mainly mitochondrial protein that is widely expressed and is highly conserved across evolution. We have previously shown that SLP-2 binds the mitochondrial lipid cardiolipin and interacts with prohibitin-1 and -2 to form specialized membrane microdomains in the mitochondrial inner membrane, which are associated with optimal mitochondrial respiration. To determine how SLP-2 functions, we performed bioenergetic analysis of primary T cells from T cell-selective Slp-2 knockout mice under conditions that forced energy production to come almost exclusively from oxidative phosphorylation. These cells had a phenotype characterized by increased uncoupled mitochondrial respiration and decreased mitochondrial membrane potential. Since formation of mitochondrial respiratory chain supercomplexes (RCS) may correlate with more efficient electron transfer during oxidative phosphorylation, we hypothesized that the defect in mitochondrial respiration in SLP-2-deficient T cells was due to deficient RCS formation. We found that in the absence of SLP-2, T cells had decreased levels and activities of complex I-III2 and I-III2-IV1-3 RCS but no defects in assembly of individual respiratory complexes. Impaired RCS formation in SLP-2-deficient T cells correlated with significantly delayed T cell proliferation in response to activation under conditions of limiting glycolysis. Altogether, our findings identify SLP-2 as a key regulator of the formation of RCS in vivo and show that these supercomplexes are required for optimal cell function. PMID:25776552

  4. 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

  5. In vivo assessment of contractile strength distinguishes differential gene function in skeletal muscle of zebrafish larvae.

    PubMed

    Martin, Brit L; Gallagher, Thomas L; Rastogi, Neha; Davis, Jonathan P; Beattie, Christine E; Amacher, Sharon L; Janssen, Paul M L

    2015-10-01

    The accessible genetics and extensive skeletal musculature of the zebrafish make it a versatile and increasingly used model for studying muscle contraction. We here describe the development of an in vivo assay for measuring the contractile force of intact zebrafish at the larval stage. In addition, as proof of applicability, we have used this assay to quantify contractile strength of zebrafish larvae in a morphant model of deranged rbfox function. Average maximum tetanic (180 Hz) whole body forces produced by wild-type larvae at 2, 3, 4, and 5 days postfertilization amounted to 3.0, 7.2, 9.1, and 10.8 mN, respectively. To compare at potentially different stages of muscle development, we developed an immunohistological assay for empirically determining the cross-sectional area of larval trunk skeletal muscle to quantify muscle-specific force per cross-sectional area. At 4-5 days postfertilization, specific force amounts to ?300 mN/mm(2), which is similar to fully developed adult mammalian skeletal muscle. We used these assays to measure contractile strength in zebrafish singly or doubly deficient for two rbfox paralogs, rbfox1l and rbfox2, which encode RNA-binding factors shown previously to modulate muscle function and muscle-specific splicing. We found rbfox2 morphants produce maximal tetanic forces similar to wild-type larvae, whereas rbfox1l morphants demonstrate significantly impaired function. rbfox1l/rbfox2 morphants are paralyzed, and their lack of contractile force production in our assay suggests that paralysis is a muscle-autonomous defect. These quantitative functional results allow measurement of muscle-specific phenotypes independent of neural input. PMID:26251513

  6. 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 ?V) 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. PMID:26026910

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

    PubMed Central

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

    2015-01-01

    A highly diverse DNA library coding for ankyrin seven-repeat proteins (ANK-N5C) was designed and constructed by a PCR-based combinatorial assembly strategy. A bacterial melibiose fermentation assay was adapted for in vivo functional screen. We isolated a transcription blocker that completely inhibits the melibiose-dependent expression of ?-galactosidase (MelA) and melibiose permease (MelB) of Escherichia coli by specifically preventing activation of the melAB operon. High-resolution crystal structural determination reveals that the designed ANK-N5C protein has a typical ankyrin fold, and the specific transcription blocker, ANK-N5C-281, forms a domain-swapped dimer. Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein. All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins. Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents. PMID:25627011

  8. In Vivo Function of PTEX88 in Malaria Parasite Sequestration and Virulence.

    PubMed

    Matz, Joachim M; Ingmundson, Alyssa; Costa Nunes, Jean; Stenzel, Werner; Matuschewski, Kai; Kooij, Taco W A

    2015-06-01

    Malaria pathology is linked to remodeling of red blood cells by eukaryotic Plasmodium parasites. Central to host cell refurbishment is the trafficking of parasite-encoded virulence factors through the Plasmodium translocon of exported proteins (PTEX). Much of our understanding of its function is based on experimental work with cultured Plasmodium falciparum, yet direct consequences of PTEX impairment during an infection remain poorly defined. Using the murine malaria model parasite Plasmodium berghei, it is shown here that efficient sequestration to the pulmonary, adipose, and brain tissue vasculature is dependent on the PTEX components thioredoxin 2 (TRX2) and PTEX88. While TRX2-deficient parasites remain virulent, PTEX88-deficient parasites no longer sequester in the brain, correlating with abolishment of cerebral complications in infected mice. However, an apparent trade-off for virulence attenuation was spleen enlargement, which correlates with a strongly reduced schizont-to-ring-stage transition. Strikingly, general protein export is unaffected in PTEX88-deficient mutants that mature normally in vitro. Thus, PTEX88 is pivotal for tissue sequestration in vivo, parasite virulence, and preventing exacerbation of spleen pathology, but these functions do not correlate with general protein export to the host erythrocyte. The presented data suggest that the protein export machinery of Plasmodium parasites and their underlying mechanistic features are considerably more complex than previously anticipated and indicate challenges for targeted intervention strategies. PMID:25820521

  9. Smoking impairs endothelial function in human saphenous vein in an ex vivo model.

    PubMed

    Sharif, M A; Bayraktutan, U; Arya, N; O'Donnell, M E; Badger, S A; Young, I S; Soong, C V

    2009-01-01

    The aim of this ex vivo experimental study was to assess the effect of smoking, diabetes mellitus, and hypertension on endothelial function in human saphenous vein, a commonly used conduit for coronary and peripheral arterial bypass surgery. A segment of long saphenous vein harvested during infrainguinal bypass surgery was mounted in an organ bath for isometric tension studies. Vein rings were precontracted to submaximal contraction with phenylephrine, followed by endothelium-dependent relaxation with acetylcholine. Long saphenous vein segments were collected from 26 patients, including five females, with a mean age of 66.4 years (range 48-92). Current smokers had impaired endothelium-dependent relaxation compared to ex- and nonsmokers (10.2%, n=13, vs. 32.9%, n=13; p<0.010). However, ex-smokers and nonsmokers did not have a significant difference in relaxant responses to acetylcholine (29.1%, n=8, vs. 24.6%, n=5; p=nonsignificant [ns]). Similarly, diabetic and nondiabetic patients did not show a significant difference in endothelium-dependent relaxation (23.1%, n=10, vs. 15.6%, n=16; p=ns). The relaxant responses in hypertensive and normotensive patients were not different (20.4%, n=12, vs. 22.5%, n=14; p=ns). Smoking has a deleterious effect on the endothelial function of saphenous vein, and smoking cessation may improve the long-term durability of saphenous vein used as a bypass graft in patients undergoing arterial reconstruction. PMID:18640818

  10. 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. PMID:26231715

  11. 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.

  12. Multiple In Vivo Biological Processes Are Mediated by Functionally Redundant Activities of Drosophila mir-279 and mir-996

    PubMed Central

    Sun, Kailiang; Jee, David; de Navas, Luis F.; Duan, Hong; Lai, Eric C.

    2015-01-01

    While most miRNA knockouts exhibit only subtle defects, a handful of miRNAs are profoundly required for development or physiology. A particularly compelling locus is Drosophila mir-279, which was reported as essential to restrict the emergence of CO2-sensing neurons, to maintain circadian rhythm, and to regulate ovarian border cells. The mir-996 locus is located near mir-279 and bears a similar seed, but they otherwise have distinct, conserved, non-seed sequences, suggesting their evolutionary maintenance for separate functions. We generated single and double deletion mutants of the mir-279 and mir-996 hairpins, and cursory analysis suggested that miR-996 was dispensable. However, discrepancies in the strength of individual mir-279 deletion alleles led us to uncover that all extant mir-279 mutants are deficient for mature miR-996, even though they retain its genomic locus. We therefore engineered a panel of genomic rescue transgenes into the double deletion background, allowing a pure assessment of miR-279 and miR-996 requirements. Surprisingly, detailed analyses of viability, olfactory neuron specification, and circadian rhythm indicate that miR-279 is completely dispensable. Instead, an endogenous supply of either mir-279 or mir-996 suffices for normal development and behavior. Sensor tests of nine key miR-279/996 targets showed their similar regulatory capacities, although transgenic gain-of-function experiments indicate partially distinct activities of these miRNAs that may underlie that co-maintenance in genomes. Altogether, we elucidate the unexpected genetics of this critical miRNA operon, and provide a foundation for their further study. More importantly, these studies demonstrate that multiple, vital, loss-of-function phenotypes can be rescued by endogenous expression of divergent seed family members, highlighting the importance of this miRNA region for in vivo function. PMID:26042831

  13. 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)…

  14. Two Forms of Chlorophyll a in vivo with Distinct Photochemical Functions Author(s): Govindjee and Eugene Rabinowitch

    E-print Network

    Govindjee

    Two Forms of Chlorophyll a in vivo with Distinct Photochemical Functions Author(s): Govindjee,u(Chlorella) and 630 m,u(Navicula) attributableto chlorophylls b and c. Thus, excitation of chlorophyll a form "chloro-700." The effect of the auxilia- ry pigments in these algae may be mediated by energy transfer to "chlorophyll

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

    PubMed Central

    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.

    2015-01-01

    Multiple sclerosis (MS) involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system (CNS). 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 (OPCs) are stem cells in the CNS and the principal source of myelinating oligodendrocytes1. OPCs are abundant in demyelinated regions of MS patients, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention2. To discover therapeutic compounds for enhancing myelination from endogenous OPCs, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem cell (EpiSC)-derived OPCs3–5. We identified seven drugs that functioned at nanomolar doses to selectively enhance the generation of mature oligodendrocytes from OPCs in vitro. Two drugs, miconazole and clobetasol, were 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 increased the number of new oligodendrocytes and enhanced remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in the experimental autoimmune encephalomyelitis (EAE) mouse model of chronic progressive MS resulted in striking reversal of disease severity. Immune response assays showed that miconazole functioned directly as a remyelinating drug with no effect on the immune system, whereas clobetasol was a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies showed that miconazole and clobetasol functioned in OPCs through mitogen-activated protein kinase (MAPK) and glucocorticoid receptor (GR) signaling, respectively. Furthermore, both drugs enhanced the generation of human oligodendrocytes from human OPCs in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally-modified derivatives, to enhance remyelination in patients. PMID:25896324

  16. Flk-1+Sca-1- mesenchymal stem cells: functional characteristics in vitro and regenerative capacity in vivo

    PubMed Central

    Li, Yugang; Pan, Enshan; Wang, Yu; Zhu, Xiaoguang; Wei, Anyang

    2015-01-01

    Objectives: Mesenchymal stem cells (MSCs) represent a powerful tool in regenerative medicine because of their differentiation and migration capacities. We aimed to investigate the possibility of Flk-1+Sca-1- mesenchymal stem cells (Flk-1+Sca-1- MSCs) transplantation to repair erectile function in patients suffering from diabetes mellitus (DM)-associated erectile dysfunction (ED). Methods: In this study, we isolated Flk-1+Sca-1- MSCs from bone marrow (bMSCs). Then, newborn male rats were intraperitoneally injected with 5-ethynyl-2-deoxyuridine for the purpose of tracking endogenous Flk-1+Sca-1- MSCs. Eight weeks later, 8 of these rats were randomly chosen to serve as normal control (N group). The remaining rats were injected intraperitoneally with 60 mg/kg of streptozotocin (STZ) to induce DM. Eight of these rats were randomly chosen to serve as DM control (DM group) while another 8 rats were subject to Flk-1+Sca-1- MSCs treatment (DM+MSC group). All rats were evaluated for erectile function by intracavernous pressure (ICP) measurement. Afterward, their penile tissues were examined by histology. Results: Flk-1+Sca-1- MSCs could differentiate into skeletal muscle cells and endothelial cells in vivo and in vitro. Engrafted Flk-1+Sca-1- MSCs were shown to home to injured muscle, participate in myofibers repair and could partially reconstitute the sarcolemmal expression of myocardin and ameliorate the level of related specific pathological markers. Conclusion: Flk-1+Sca-1- MSCs could be used in the treatment erectile function in diabetes mellitus associated erectile dysfunction by promoting regeneration of nNOS-positive nerves, endothelium, and smooth muscle in the penis.

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

    PubMed Central

    Schick, Martin Alexander; Baar, Wolfgang; Bruno, Raphael Romano; Wollborn, Jakob; Held, Christopher; Schneider, Reinhard; Flemming, Sven; Schlegel, Nicolas; Roewer, Norbert; Neuhaus, Winfried; Wunder, Christian

    2015-01-01

    Volume therapy is a standard procedure in daily perioperative care, and there is an ongoing discussion about the benefits of colloid resuscitation with hydroxyethylstarch (HES). In sepsis HES should be avoided due to a higher risk for acute kidney injury (AKI). Results of the usage of HES in patients without sepsis are controversial. Therefore we conducted an animal study to evaluate the impact of 6% HES 130/0.4 on kidney integrity with sepsis or under healthy conditions Sepsis was induced by standardized Colon Ascendens Stent Peritonitis (sCASP). sCASP-group as well as control group (C) remained untreated for 24 h. After 18 h sCASP+HES group (sCASP+VOL) and control+HES (C+VOL) received 50 ml/KG balanced 6% HES (VOL) 130/0.4 over 6h. After 24h kidney function was measured via Inulin- and PAH-Clearance in re-anesthetized rats, and serum urea, creatinine (crea), cystatin C and Neutrophil gelatinase-associated lipocalin (NGAL) as well as histopathology were analysed. In vitro human proximal tubule cells (PTC) were cultured +/- lipopolysaccharid (LPS) and with 0.1–4.0% VOL. Cell viability was measured with XTT-, cell toxicity with LDH-test. sCASP induced severe septic AKI demonstrated divergent results regarding renal function by clearance or creatinine measure focusing on VOL. Soleley HES (C+VOL) deteriorated renal function without sCASP. Histopathology revealed significantly derangements in all HES groups compared to control. In vitro LPS did not worsen the HES induced reduction of cell viability in PTC cells. For the first time, we demonstrated, that application of 50 ml/KG 6% HES 130/0.4 over 6 hours induced AKI without inflammation in vivo. Severity of sCASP induced septic AKI might be no longer susceptible to the way of volume expansion. PMID:26340751

  18. 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 signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients. PMID:25896324

  19. 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

  20. Functional data analysis: classification and regression 

    E-print Network

    Lee, Ho-Jin

    2005-11-01

    Functional data refer to data which consist of observed functions or curves evaluated at a finite subset of some interval. In this dissertation, we discuss statistical analysis, especially classification and regression ...

  1. In-vivo corneal biomechanical analysis of unilateral keratoconus

    PubMed Central

    Ayar, Orhan; Ozmen, Mehmet Cuneyt; Muftuoglu, Orkun; Akdemir, Mehmet Orcun; Koc, Mustafa; Ozulken, Kemal

    2015-01-01

    AIM To evaluate and compare corneal biomechanical findings measured by ocular response analyzer, topographic and pachymetric findings in patients with unilateral keratoconus patients and healthy controls. METHODS This is an observational, case-control study. Patients with keratoconus in one eye and forme fruste keratoconus in the fellow eye were compared with sex and age matched with controls healthy subjects. All subjects were evaluated with rotating scheimpflug imaging system. The receiver-operating-characteristic curves were analyzed to evaluate the sensitivity and specificity of the parameters. RESULTS Twenty-seven patients with keratoconus in one eye and forme fruste keratoconus in the fellow eye were compared with 40 eyes of 40 normal subjects. Corneal hysteresis (CH) was 8.0±1.7 mm Hg in keratoconus group, 8.3±1.6 mm Hg in forme fruste keratoconus group, and 9.8±1.6 mm Hg in control groups (P=0.54 between keratoconus and forme fruste keratoconus groups, P<0.01 between control group and other groups). Corneal resistance factor (CRF) was 7.1±2.2 mm Hg in keratoconus group, 7.8±1.2 mm Hg in forme fruste keratoconus group and 9.9±1.5 mm Hg in control group (P<0.001 between control group and other groups). Using receiver-operating-characteristic analysis, the area under curve values of the parameters to distinguish forme fruste keratoconus from control subjects were: CH (0.768), CRF (0.866). Best cut-off points were 9.3 mm Hg and 8.8 mm Hg for CH and CRF respectively. CONCLUSION Ocular response analyzer parameters (CH and CRF) are found to be significantly lower in forme fruste keratoconus patients compared to normal control subjects. PMID:26682162

  2. 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/,/ř/,/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.

  3. 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

  4. Functionalization of iron oxide magnetic nanoparticles with targeting ligands: their physicochemical properties and in vivo behavior

    PubMed Central

    Fang, Chen; Veiseh, Omid; Kievit, Forrest; Bhattarai, Narayan; Wang, Freddy; Stephen, Zach; Li, Chun; Lee, Donghoon; Ellenbogen, Richard G.; Zhang, Miqin

    2010-01-01

    Aims To develop and evaluate two tumor-specific nanoprobes by functionalization of a PEG-immobilized nanoparticle with arginine-glycine-aspartic acid (RGD) or chlorotoxin (CTX) ligand that targets ?v?3 integrin and MMP-2 receptors, respectively. Materials and Methods The nanoprobes were made of iron oxide cores, biocompatible polymer coating, and surface-conjugated RGD or CTX peptide. The tumor-targeting specificity of the nanoprobes was evaluated both in vitro and in vivo. Results and Discussion Both nanoprobes were highly dispersive and exhibited excellent long-term stability in cell culture media. The RGD-conjugated nanoprobe displayed a strong initial accumulation near neovasculatures in tumors followed by quick clearance. Conversely, the CTX-enabled nanoprobe exhibited sustained accumulation throughout the tumor. Conclusion These findings revealed the influence of the targeting ligands on the intratumoral distribution of the ligand-enabled nanoprobes. With flexible surface chemistry, our nanoparticle platform can be used in a modular fashion to conjugate biomolecules for intended applications. PMID:21128719

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

    PubMed

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

    2015-10-01

    The accumulation of DNA and RNA oxidative damage is observed in cortical and hippocampal neurons from Alzheimer's disease (AD) brains at early stages of pathology. We recently reported that Tau is a key nuclear player in the protection of neuronal nucleic acid integrity in vivo under physiological conditions and hyperthermia, a strong inducer of oxidative stress. In a mouse model of tauopathy (THY-Tau22), we demonstrate that hyperthermia selectively induces nucleic acid oxidative damage and nucleic acid strand breaks in the nucleus and cytoplasm of hippocampal neurons that display early Tau phosphorylation but no Tau fibrils. Nucleic acid-damaged neurons were exclusively immunoreactive for prefibrillar Tau oligomers. A similar association between prefibrillar Tau oligomers and nucleic acid oxidative damage was observed in AD brains. Pretreatment with Methylene Blue (MB), a Tau aggregation inhibitor and a redox cycler, reduced hyperthermia-induced Tau oligomerization as well as nucleic acid damage. This study clearly highlights the existence of an early and critical time frame for hyperthermia-induced Tau oligomerization, which most likely occurs through increased oxidative stress, and nucleic acid vulnerability during the progression of Tau pathology. These results suggest that at early stages of AD, Tau oligomerization triggers the loss of the nucleic acid protective function of monomeric Tau. This study highlights the existence of a short therapeutic window in which to prevent the formation of pathological forms of Tau and their harmful consequences on nucleic acid integrity during the progression of Tau pathology. PMID:26385829

  6. 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. PMID:24445121

  7. Glycan variants of a respiratory syncytial virus antibody with enhanced effector function and in vivo efficacy

    PubMed Central

    Hiatt, Andrew; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do; Pauly, Michael H.; Velasco, Jesus; Whaley, Kevin J.; Piedra, Pedro A.; Gilbert, Brian E.; Zeitlin, Larry

    2014-01-01

    Respiratory syncytial virus (RSV) can cause devastating lower respiratory tract infections in preterm infants or when other serious health problems are present. Immunoprophylaxis with palivizumab (Synagis), a humanized IgG1 mAb, is the current standard of care for preventing RSV infection in at-risk neonates. We have explored the contribution of effector function to palivizumab efficacy using a plant-based expression system to produce palivizumab N-glycan structure variants with high homogeneity on different antibody isotypes. We compared these isotype and N-glycoform variants with commercially available palivizumab with respect to both in vitro receptor and C1q binding and in vivo efficacy. Whereas the affinity for antigen and neutralization activity of each variant were indistinguishable from those of palivizumab, their Fc? receptor binding profiles were very different, which was reflected in either a reduced or enhanced ability to influence the RSV lung titer in challenged cotton rats. Enhanced Fc? receptor binding was associated with reduced viral lung titers compared with palivizumab, whereas abrogation of receptor binding led to a drastic reduction in efficacy. The results support the hypotheses that classic antibody neutralization is a minor component of efficacy by palivizumab in the cotton rat and that antibody-dependent cell-mediated cytotoxicity activity can significantly enhance the efficacy of this antiviral mAb. PMID:24711420

  8. Functional and fine structural changes in isolated rat lungs challenged with endotoxin ex vivo and in vitro.

    PubMed Central

    Uhlig, S.; Brasch, F.; Wollin, L.; Fehrenbach, H.; Richter, J.; Wendel, A.

    1995-01-01

    The aim of this study was to relate changes in rat lung functions caused by the endotoxin lipopolysaccharide (LPS) to alterations in structure. The following four experimental groups were used: 1), control in vitro, perfusion for 150 minutes; 2), LPS in vitro, perfusion for 150 minutes and infusion of 5 mg of LPS after 40 minutes; 3), control ex vivo, perfusion for 10 minutes; and 4), LPS ex vivo, lungs perfused for 10 minutes from rats treated for 110 minutes with 20 mg/kg LPS intraperitoneally. Histologically, blood-derived leukocytes were detectable only in lungs from group 4, where neutrophils were found in capillaries, interstitium, and endothelial pouches. LPS treatment increased pulmonary resistance and decreased pulmonary compliance in group 4 (ex vivo), and, to a greater extent, in group 2 (in vitro). In these two groups, formation of giant lamellar bodies in the type II pneumocytes was observed. By histological examination, the bronchoconstriction induced by LPS in vitro was localized to the terminal bronchioles. At 2 hours after LPS treatment, no edema and no change in precapillary and postcapillary resistance, capillary pressure, vascular compliance, capillary permeability, and the wet/dry ratio was observed. Thus, our major findings are that LPS induced constriction of the terminal bronchioles in vitro, formation of giant lamellar bodies in type II pneumocytes ex vivo and in vitro, and trapping of neutrophils in endothelial pouches in vivo. Images Figure 2 Figure 3 Figure 4 Figure 6 PMID:7747816

  9. 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 concentrations constitutes environmental cardiovascular risk factor.

  10. Resveratrol and diabetic cardiac function: focus on recent in vitro and in vivo studies.

    PubMed

    Turan, Belma; Tuncay, Erkan; Vassort, Guy

    2012-04-01

    Resveratrol, a natural phytoalexin found in wine has the potential to impact a variety of human diseases. Resveratrol like other polyphenols activates many of the same intracellular pathways as those activated by caloric restriction. It can quench reactive oxidative species, ROS and induce eNOS and iNOS expression. Resveratrol also can activate SIRT1, a NAD?-dependent deacetylase, that leads an improved in mitochondrial function, and then this procedure turns to activate the transcription factor Nrf2 that coordinates expression of key antioxidant mechanisms by binding to the antioxidant response elements. Resveratrol provides cardioprotection by triggering preconditioning and inducing autophagy. It also presents chemical similarities with estrogen and was reported to activate both nuclear and extranuclear estrogen receptors. Resveratrol treatment alleviated diabetes-induced cardiovascular system disorders via different endogeneous signaling pathways including oxidative stress/antioxidant defense system, glucose/insulin metabolism, overexpression of iNOS/nitrotyrosine, and preconditioning. Resveratrol treatment significantly reduced the blood glucose level in STZ-treated type 1 diabetic animals through insulin-dependent and insulin-independent pathways. Resveratrol triggers some of the similar intracellular insulin signalling components in myocardium such as eNOS, AKT through the AMPK pathway, and plays an essential role in Glut-4 translocation and glucose uptake in STZ-induced diabetic myocardium. However, resveratrol can exhibit hormetic action expressing health benefits at lower doses whereas being detrimental at higher doses. It might also exert antidiabetic effects by activating SIRT1 directly in the brain. This review includes a summary of the role of resveratrol and diabetic cardiac function including a brief discussion on in vitro and in vivo studies as well as our original observations in diabetic rats. PMID:22437738

  11. In Vivo Characterization of Traumatic Brain Injury Neuropathology with Structural and Functional Neuroimaging

    PubMed Central

    LEVINE, BRIAN; FUJIWARA, ESTHER; O’CONNOR, CHARLENE; RICHARD, NADINE; KOVACEVIC, NATASA; MANDIC, MARINA; RESTAGNO, ADRIANA; EASDON, CRAIG; ROBERTSON, IAN H.; GRAHAM, SIMON J.; CHEUNG, GORDON; GAO, FUQIANG; SCHWARTZ, MICHAEL L.; BLACK, SANDRA E.

    2007-01-01

    Quantitative neuroimaging is increasingly used to study the effects of traumatic brain injury (TBI) on brain structure and function. This paper reviews quantitative structural and functional neuroimaging studies of patients with TBI, with an emphasis on the effects of diffuse axonal injury (DAI), the primary neuropathology in TBI. Quantitative structural neuroimaging has evolved from simple planometric measurements through targeted region-of-interest analyses to whole-brain analysis of quantified tissue compartments. Recent studies converge to indicate widespread volume loss of both gray and white matter in patients with moderate-to-severe TBI. These changes can be documented even when patients with focal lesions are excluded. Broadly speaking, performance on standard neuropsychological tests of speeded information processing are related to these changes, but demonstration of specific brain-behavior relationships requires more refined experimental behavioral measures. The functional consequences of these structural changes can be imaged with activation functional neuroimaging. Although this line of research is at an early stage, results indicate that TBI causes a more widely dispersed activation in frontal and posterior cortices. Further progress in analysis of the consequences of TBI on neural structure and function will require control of variability in neuropathology and behavior. PMID:17020478

  12. 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 ?g/mL ICG and 60 g/L albumin. ICG fluorescence can be detected at a concentration of 150 ?g/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.

  13. Structural domains required for Caenorhabditis elegans G protein-coupled receptor kinase 2 (GRK-2) function in vivo.

    PubMed

    Wood, Jordan F; Wang, Jianjun; Benovic, Jeffrey L; Ferkey, Denise M

    2012-04-13

    G protein-coupled receptor kinases (GRKs) are key regulators of signal transduction that specifically phosphorylate activated G protein-coupled receptors (GPCRs) to terminate signaling. Biochemical and crystallographic studies have provided great insight into mammalian GRK2/3 interactions and structure. However, despite extensive in vitro characterization, little is known about the in vivo contribution of these described GRK structural domains and interactions to proper GRK function in signal regulation. We took advantage of the disrupted chemosensory behavior characteristic of Caenorhabditis elegans grk-2 mutants to discern the interactions required for proper in vivo Ce-GRK-2 function. Informed by mammalian crystallographic and biochemical data, we introduced amino acid substitutions into the Ce-grk-2 coding sequence that are predicted to selectively disrupt GPCR phosphorylation, G?(q/11) binding, G?? binding, or phospholipid binding. Changing the most amino-terminal residues, which have been shown in mammalian systems to be required specifically for GPCR phosphorylation but not phosphorylation of alternative substrates or recruitment to activated GPCRs, eliminated the ability of Ce-GRK-2 to restore chemosensory signaling. Disrupting interaction between the predicted Ce-GRK-2 amino-terminal ?-helix and kinase domain, posited to stabilize GRKs in their active ATP- and GPCR-bound conformation, also eliminated Ce-GRK-2 chemosensory function. Finally, although changing residues within the RH domain, predicted to disrupt interaction with G?(q/11), did not affect Ce-GRK-2 chemosensory function, disruption of the predicted PH domain-mediated interactions with G?? and phospholipids revealed that both contribute to Ce-GRK-2 function in vivo. Combined, we have demonstrated functional roles for broadly conserved GRK2/3 structural domains in the in vivo regulation of organismal behavior. PMID:22375004

  14. 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.

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

    PubMed Central

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

    2012-01-01

    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. PMID:22380131

  16. 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.

  17. 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

  18. 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.

  19. 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…

  20. Set1 and MLL1/2 Target Distinct Sets of Functionally Different Genomic Loci In Vivo.

    PubMed

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

    2015-12-29

    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

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

    PubMed Central

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

    2007-01-01

    AIM: To evaluate a newly developed hand-held confocal probe for in vivo microscopic imaging of the complete gastrointestinal tract in rodents. METHODS: A novel rigid confocal probe (diameter 7 mm) was designed with optical features similar to the flexible endomicroscopy system for use in humans using a 488 nm single line laser for fluorophore excitation. Light emission was detected at 505 to 750 nm. The field of view was 475 ?m × 475 ?m. Optical slice thickness was 7 ?m with a lateral resolution of 0.7 ?m. Subsurface serial images at different depths (surface to 250 ?m) were generated in real time at 1024 × 1024 pixels (0.8 frames/s) by placing the probe onto the tissue in gentle, stable contact. Tissue specimens were sampled for histopathological correlation. RESULTS: The esophagus, stomach, small and large intestine and meso, liver, pancreas and gall bladder were visualised in vivo at high resolution in n = 48 mice. Real time microscopic imaging with the confocal mini-microscopy probe was easy to achieve. The different staining protocols (fluorescein, acriflavine, FITC-labelled dextran and L. esculentum lectin) each highlighted specific aspects of the tissue, and in vivo imaging correlated excellently with conventional histology. In vivo blood flow monitoring added a functional quality to morphologic imaging. CONCLUSION: Confocal microscopy is feasible in vivo allowing the visualisation of the complete GI tract at high resolution even of subsurface tissue structures. The new confocal probe design evaluated in this study is compatible with laparoscopy and significantly expands the field of possible applications to intra-abdominal organs. It allows immediate testing of new in vivo staining and application options and therefore permits rapid transfer from animal studies to clinical use in patients. PMID:17465494

  2. 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.

  3. Enzymatic Characterization and In Vivo Function of Five Terminal Oxidases in Pseudomonas aeruginosa

    PubMed Central

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

    2014-01-01

    The ubiquitous opportunistic pathogen Pseudomonas aeruginosa has five aerobic terminal oxidases: bo3-type quinol oxidase (Cyo), cyanide-insensitive oxidase (CIO), aa3-type cytochrome c oxidase (aa3), and two cbb3-type cytochrome c oxidases (cbb3-1 and cbb3-2). 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 cbb3-1 and cbb3-2, indicating that Cyo, CIO, and aa3 are low-affinity enzymes and that cbb3-1 and cbb3-2 are high-affinity enzymes. Although cbb3-1 and cbb3-2 exhibited different expression patterns in response to oxygen concentration, they had similar Km values for oxygen. Both cbb3-1 and cbb3-2 utilized cytochrome c4 as the main electron donor under normal growth conditions. The electron transport chains terminated by cbb3-1 and cbb3-2 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. PMID:25182500

  4. In vivo skin biophysical behaviour and surface topography as a function of ageing.

    PubMed

    Pailler-Mattei, C; Debret, R; Vargiolu, R; Sommer, P; Zahouani, H

    2013-12-01

    Normal skin ageing is characterised by an alteration of the underlying connective tissue with measurable consequences on global skin biophysical properties. The cutis laxa syndrome, a rare genetic disorder, is considered as an accelerated ageing process since patients appear prematurely aged due to alterations of dermal elastic fibres. In the present study, we compared the topography and the biomechanical parameters of normal aged skin with an 17 year old cutis laxa patient. Skin topography analyses were conducted on normal skin at different ages. The results indicate that the skin relief highly changes as a function of ageing. The cutaneous lines change from a relatively isotropic orientation to a highly anisotropic orientation. This reorganisation of the skin relief during the ageing process might be due to a modification of the skin mechanical properties, and particularly to a modification of the dermis mechanical properties. A specific bio-tribometer, based on the indentationtechnique under light load, has been developed to study the biophysical properties of the human skin in vivo through two main parameters: the physico-chemical properties of the skin surface, by measuring the maximum adhesion force between the skin and the bio-tribometer; and the bulk mechanical properties. Our results show that the pull-off force between the skin and the biotribometer as well as the skin Young's modulus decrease with age. In the case of the young cutis laxa patient, the results obtained were similar to those observed for aged individuals. These results are very interesting and encouraging since they would allow the monitoring of the cutis laxa skin in a standardised and non-invasive way to better characterize either the evolution of the disease or the benefit of a treatment. PMID:23664827

  5. 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

  6. Error analysis for the in-vivo measurement of radionuclides in wounds: Monte Carlo study.

    PubMed

    Ahmed, A S M Sabbir; Capello, Kevin; Sabourin, Trevor; Kramer, Gary H

    2010-12-01

    This paper describes calculation of error associated with the direct in-vivo measurements of radionuclides in a wound. A typical radiation injury to a hand with Am radionuclide is illustrated for error analysis. A Monte Carlo model was developed and the detector pulse spectrum studied with a custom-designed HPGe detector. A pinhole collimator was designed, and its performance with a wide area detector was studied. The results show that significant errors might propagate if the lowest energy peaks of Am are used during in vivo measurements of the wound. In comparison to that, less uncertainty was found for 26.3 and 59.5 keV gamma peaks, and those levels are recommended for estimation of wound depth and activity. PMID:21068594

  7. 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; Valentăo, 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, it evidences zebrafish's potential for in vivo efficacy or toxicity screening of ubiquinone analogues or antiparasitic mitochondria-targeted drugs. PMID:23758163

  8. Assessment of blood clot formation and platelet receptor function ex vivo in patients with primary Sjögren's syndrome

    PubMed Central

    Collins, K S; Balasubramaniam, K; Viswanathan, G; Natasari, A; Tarn, J; Lendrem, D; Mitchell, S; Zaman, A; Ng, W F

    2013-01-01

    Objectives Primary Sjögren's syndrome (pSS) shares clinical features and pathogenetic mechanisms with systemic lupus erythematosus (SLE). SLE is associated with an increased thromboembolic risk; however, it is unclear whether pSS patients are susceptible to thromboembolic diseases. In this study, we examined ex vivo blood clot formation (clot strength, rates of clot formation and lysis) in pSS using thromboelastography (TEG) and platelet aggregation to common agonists using multiple electrode aggregometry (MEA). We also investigated the relationship between TEG/MEA parameters and clinical/laboratory features of pSS. Design Case control. Setting Secondary care, single centre. Participants 34 pSS patients, 11 SLE patients and 13 healthy volunteers (all women) entered and completed the study. Primary and secondary outcome measures Primary outcomes: TEG and MEA parameters between three subject groups. Secondary outcomes: The relationships between TEG/MEA and clinical/laboratory parameters analysed using bivariate correlation analysis with corrections for multiple testing. Results All TEG and MEA parameters were similar for the three subject groups. After corrections for multiple testing, interleukin (IL)-1? and Macrophage inflammatory proteins (MIP)-1? remain correlated inversely with clot strength (r=?0.686, p=0.024 and r=?0.730, p=0.012, respectively) and overall coagulability (r=?0.640, p=0.048 and r=?0.648, p=0.048). Stepwise regression analysis revealed that several cytokines such as MIP-1?, IL-17a, IL-1? and Interferon (IFN)-? may be key predictors of clot strength and overall coagulability in pSS. Conclusions Clot kinetics and platelet receptor function are normal in pSS. Several cytokines correlate with clot strength and overall coagulability in pSS. PMID:23793707

  9. 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.

  10. 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.

  11. In vivo image analysis of BoHV-4-based vector in mice.

    PubMed

    Franceschi, Valentina; Stellari, Fabio Franco; Mangia, Carlo; Jacca, Sarah; Lavrentiadou, Sophia; Cavirani, Sandro; Heikenwalder, Mathias; Donofrio, Gaetano

    2014-01-01

    Due to its biological characteristics bovine herpesvirus 4 (BoHV-4) has been considered as an appropriate gene delivery vector. Its genomic clone, modified as a bacterial artificial chromosome (BAC), is better genetically manipulable and can be used as an efficient gene delivery and vaccine vector. Although a large amount of data have been accumulated in vitro on this specific aspect, the same cannot be asserted for the in vivo condition. Therefore, here we investigated the fate of a recombinant BoHV-4 strain expressing luciferase (BoHV-4-A-CMVluc?TK) after intraperitoneal or intravenous inoculation in mice, by generating a novel recombinant BoHV-4 expressing luciferase (BoHV-4-A-CMVluc?TK) and by following the virus replication through in vivo imaging analysis. BoHV-4-A-CMVluc?TK was first characterized in vitro where it was shown, on one hand that its replication properties are identical to those of the parental virus, and on the other that the transduced/infected cells strongly express luciferase. When BoHV-4-A-CMVluc?TK was inoculated in mice, either intraperitoneally or intravenously, BoHV-4-A-CMVluc?TK infection/transduction was exclusively localized to the liver, as detected by in vivo image analysis, and in particular almost exclusively in the hepatocytes, as determined by immuno-histochemistry. These data, that add a new insight on the biology of BoHV-4 in vivo, provide the first indication for the potential use of a BoHV-4-based vector in gene-transfer in the liver. PMID:24752229

  12. In vivo stem cell function of interleukin-3-induced blast cells

    SciTech Connect

    Tsunoda, J.; Okada, S.; Suda, J.; Nagayoshi, K.; Nakauchi, H.; Hatake, K.; Miura, Y.; Suda, T. )

    1991-07-15

    The treatment of mice with high doses of 5-fluorouracil (5-FU) results in an enrichment of primitive hematopoietic progenitors. Using this procedure, the authors obtained a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro and could differentiate into not only myeloid cells but also into lymphoid lineage cells. The phenotypes of interleukin-3 (IL-3) induced blast colony cells were Thy-1-positive and lineage-marker-negative. They examined whether these blast colony cells contained primitive hematopoietic stem cells in vivo and could reconstitute hematopoietic tissues in lethally irradiated mice. Blast colony cells could generate macroscopic visible spleen colonies on days 8 and 12, and 5 {times} 10(3) blast cells were sufficient to protect them from lethally irradiation. It was shown that 6 or 8 weeks after transplantation of 5 {times} 10(3) blast cells, donor male cells were detected in the spleen and thymus of the female recipients but not in the bone marrow by Southern blot analysis using Y-encoded DNA probe. After 10 weeks, bone marrow cells were partially repopulated from donor cells. In a congenic mouse system, donor-derived cells (Ly5.2) were detected in the thymus and spleen 6 weeks after transplantation. Fluorescence-activated cell sorter analyses showed that B cells and macrophages developed from donor cells in the spleen. In the thymus, donor-derived cells were found in CD4, CD8 double-positive, single-positive, and double-negative populations. Reconstitution of bone marrow was delayed and myeloid and lymphoid cells were detected 10 weeks after transplantation. These results indicate that IL-3-induced blast cells contain the primitive hematopoietic stem cells capable of reconstituting hematopoietic organs in lethally irradiated mice.

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

    PubMed Central

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

    2012-01-01

    Background The polarized reorganization of the T cell membrane and intracellular signaling molecules in response to T cell receptor (TCR) engagement has been implicated in the modulation of T cell development and effector responses. In siRNA-based studies Dlg1, a MAGUK scaffold protein and member of the Scribble polarity complex, has been shown to play a role in T cell polarity and TCR signal specificity, however the role of Dlg1 in T cell development and function in vivo remains unclear. Methodology/Principal Findings Here we present the combined data from three independently-derived dlg1-knockout mouse models; two germline deficient knockouts and one conditional knockout. While defects were not observed in T cell development, TCR-induced early phospho-signaling, actin-mediated events, or proliferation in any of the models, the acute knockdown of Dlg1 in Jurkat T cells diminished accumulation of actin at the IS. Further, while Th1-type cytokine production appeared unaffected in T cells derived from mice with a dlg1germline-deficiency, altered production of TCR-dependent Th1 and Th2-type cytokines was observed in T cells derived from mice with a conditional loss of dlg1 expression and T cells with acute Dlg1 suppression, suggesting a differential requirement for Dlg1 activity in signaling events leading to Th1 versus Th2 cytokine induction. The observed inconsistencies between these and other knockout models and siRNA strategies suggest that 1) compensatory upregulation of alternate gene(s) may be masking a role for dlg1 in controlling TCR-mediated events in dlg1 deficient mice and 2) the developmental stage during which dlg1 ablation begins may control the degree to which compensatory events occur. Conclusions/Significance These findings provide a potential explanation for the discrepancies observed in various studies using different dlg1-deficient T cell models and underscore the importance of acute dlg1 ablation to avoid the upregulation of compensatory mechanisms for future functional studies of the Dlg1 protein. PMID:23028902

  14. 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

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

    PubMed

    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. PMID:26220210

  16. 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.

  17. 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.

  18. Surface Based Analysis of Diffusion Orientation for Identifying Architectonic Domains in the In Vivo Human Cortex

    PubMed Central

    McNab, Jennifer A.; Polimeni, Jonathan R.; Wang, Ruopeng; Augustinack, Jean C.; Fujimoto, Kyoko; Player, Allison; Janssens, Thomas; Farivar, Reza; Folkerth, Rebecca D.; Vanduffel, Wim; Wald, Lawrence L.

    2012-01-01

    Diffusion tensor MRI is sensitive to the coherent structure of brain tissue and is commonly used to study large-scale white matter structure. Diffusion in grey matter is more isotropic, however, several groups have observed coherent patterns of diffusion anisotropy within the cerebral cortical grey matter. We extend the study of cortical diffusion anisotropy by relating it to the local coordinate system of the folded cerebral cortex. We use 1mm and sub-millimeter isotropic resolution diffusion imaging to perform a laminar analysis of the principal diffusion orientation, fractional anisotropy, mean diffusivity and partial volume effects. Data from 6 in vivo human subjects, a fixed human brain specimen and an anesthetized macaque were examined. Large regions of cortex show a radial diffusion orientation. In vivo human and macaque data displayed a sharp transition from radial to tangential diffusion orientation at the border between primary motor and somatosensory cortex, and some evidence of tangential diffusion in secondary somatosensory cortex and primary auditory cortex. Ex vivo diffusion imaging in a human tissue sample showed some tangential diffusion orientation in S1 but mostly radial diffusion orientations in both M1 and S1. PMID:23247190

  19. 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

  20. In Vivo Risk Analysis of Pancreatic Cancer Through Optical Characterization of Duodenal Mucosa

    PubMed Central

    Mutyal, Nikhil N.; Radosevich, Andrew J.; Bajaj, Shailesh; Konda, Vani; Siddiqui, Uzma D.; Waxman, Irving; Goldberg, Michael J.; Rogers, Jeremy D.; Gould, Bradley; Eshein, Adam; Upadhye, Sudeep; Koons, Ann; Gonzalez-Haba Ruiz, Mariano; Roy, Hemant K.; Backman, Vadim

    2015-01-01

    Objectives To reduce pancreatic cancer mortality, a paradigm shift in cancer screening is needed. Our group pioneered the use of low-coherence enhanced backscattering (LEBS) spectroscopy to predict the presence of pancreatic cancer by interrogating the duodenal mucosa. A previous ex vivo study (n = 203) demonstrated excellent diagnostic potential: sensitivity, 95%; specificity, 71%; and accuracy, 85%. The objective of the current case-control study was to evaluate this approach in vivo. Methods We developed a novel endoscope-compatible fiber-optic probe to measure LEBS in the periampullary duodenum of 41 patients undergoing upper endoscopy. This approach enables minimally invasive detection of the ultrastructural consequences of pancreatic field carcinogenesis. Results The LEBS parameters and optical properties were significantly altered in patients harboring adenocarcinomas (including early-stage) throughout the pancreas relative to healthy controls. Test performance characteristics were excellent with sensitivity = 78%, specificity = 85%, and accuracy = 81%. Moreover, the LEBS prediction rule was not confounded by patients’ demographics. Conclusion We demonstrate the feasibility of in vivo measurement of histologically normal duodenal mucosa to predict the presence of adenocarcinoma throughout the pancreas. This represents the next step in establishing duodenal LEBS analysis as a prescreening technique that identifies clinically asymptomatic patients who are at elevated risk of PC. PMID:25906443

  1. FRATS: Functional Regression Analysis of DTI Tract Statistics.

    PubMed

    Zhu, Hongtu; Styner, Martin; Tang, Niansheng; Liu, Zhexing; Lin, Weili; Gilmore, John H

    2010-04-01

    Diffusion tensor imaging (DTI) provides important information on the structure of white matter fiber bundles as well as detailed tissue properties along these fiber bundles in vivo. This paper presents a functional regression framework, called FRATS, for the analysis of multiple diffusion properties along fiber bundle as functions in an infinite dimensional space and their association with a set of covariates of interest, such as age, diagnostic status and gender, in real applications. The functional regression framework consists of four integrated components: the local polynomial kernel method for smoothing multiple diffusion properties along individual fiber bundles, a functional linear model for characterizing the association between fiber bundle diffusion properties and a set of covariates, a global test statistic for testing hypotheses of interest, and a resampling method for approximating the p-value of the global test statistic. The proposed methodology is applied to characterizing the development of five diffusion properties including fractional anisotropy, mean diffusivity, and the three eigenvalues of diffusion tensor along the splenium of the corpus callosum tract and the right internal capsule tract in a clinical study of neurodevelopment. Significant age and gestational age effects on the five diffusion properties were found in both tracts. The resulting analysis pipeline can be used for understanding normal brain development, the neural bases of neuropsychiatric disorders, and the joint effects of environmental and genetic factors on white matter fiber bundles. PMID:20335089

  2. 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 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. Electronic supplementary information (ESI) available: Experimental details on the preparation of HNO3 functionalized CNTs and supplementary analyses (?-Raman, TG, EDS, acid-base titration, FTIR, roughness measurements, SEM and optical images) are shown. See DOI: 10.1039/c5nr01829c

  3. In Vitro and In Vivo Studies of Single-Walled Carbon Nanohorns with Encapsulated Metallofullerenes and Exohedrally Functionalized Quantum Dots

    SciTech Connect

    Zhang, Jianfei; Ge, Jiechao; Shultz, M.D.; Chung, Eunna; Singh, Gurpreet; Shu, Chunying; Deck, Paul; Fatouros, Panos; Henderson, Scott; Corwin, Frank; Geohegan, David B; Rouleau, Christopher M; More, Karren Leslie; Rylander, Nichole M; Rylander, Christopher; Gibson, Harry W; Dorn, Harry C

    2010-07-01

    Single-walled carbon nanohorns (SWNHs) are new carbonaceous materials. In this paper, we report the first successful preparation of SWNHs encapsulating trimetallic nitride template endohedral metallofullerenes (TNT-EMFs). The resultant materials were functionalized by a high-speed vibration milling method and conjugated with CdSe/ZnS quantum dots (QDs). The successful encapsulation of TNT-EMFs and external functionalization with QDs provide a dual diagnostic platform for in vitro and in vivo biomedical applications of these new carbonaceous materials.

  4. 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. PMID:26712849

  5. 3-D in vivo brain tumor geometry study by scaling analysis

    NASA Astrophysics Data System (ADS)

    Torres Hoyos, F.; Martín-Landrove, M.

    2012-02-01

    A new method, based on scaling analysis, is used to calculate fractal dimension and local roughness exponents to characterize in vivo 3-D tumor growth in the brain. Image acquisition was made according to the standard protocol used for brain radiotherapy and radiosurgery, i.e., axial, coronal and sagittal magnetic resonance T1-weighted images, and comprising the brain volume for image registration. Image segmentation was performed by the application of the k-means procedure upon contrasted images. We analyzed glioblastomas, astrocytomas, metastases and benign brain tumors. The results show significant variations of the parameters depending on the tumor stage and histological origin.

  6. 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.

  7. Spectral analysis of photo-induced delayed luminescence from human skin in vivo

    NASA Astrophysics Data System (ADS)

    Musumeci, Francesco; Lanzanň, Luca; Privitera, Simona; Tudisco, Salvatore; Scordino, Agata

    2007-07-01

    The UVA induced Delayed Luminescence (DL), has been measured in vivo in the forearm skin of some healthy volunteers of different sex and age during several periods of the year. An innovative instrument able to detect, in single photon counting mode, the spectrum and the time trend of the DL emission has been used. The measured differences in the time trends of the spectral components may be related to the sex and the age. The potential development of a new analysis technique based on this phenomenon is discussed.

  8. Ex Vivo Expansion of Functional Human UCB-HSCs/HPCs by Coculture with AFT024-hkirre Cells

    PubMed Central

    Khan, Muti ur Rehman; Ali, Ijaz; Jiao, Wei; Wang, Yun; Masood, Saima; Yousaf, Muhammad Zubair; Javaid, Aqeel; Ahmad, Shafique; Feng, Meifu

    2014-01-01

    Kiaa1867 (human Kirre, hKirre) has a critical role in brain development and/or maintenance of the glomerular slit diaphragm in kidneys. Murine homolog of this gene, mKirre expressed in OP9 and AFT024 cells could support hematopoietic stem cells/hematopoietic progenitor cells (HSC/HPC) expansion in vitro. HKirre is also expressed in human FBMOB-hTERT cell line and fetal liver fibroblast-like cells but its function has remained unclear. In this paper, we cloned a hKirre gene from human fetal liver fibroblast-like cells and established a stably overexpressing hKirre-AFT024 cell line. Resultant cells could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than AFT024-control cells transformed with mock plasmid. The Expanded human umbilical cord blood (hUCB) CD34+ cells retained the capacity of multipotent differentiation as long as 8 weeks and successfully repopulated the bone marrow of sublethally irradiated NOD/SCID mice, which demonstrated the expansion of long-term primitive transplantable HSCs/HPCs. Importantly, hkirre could upregulate the expressions of Wnt-5A, BMP4, and SDF-1 and downregulate TGF-? with other hematopoietic growth factors. By SDS-PAGE and Western Blot analysis, a ~89?kDa protein in total lysate of AFT024-hKirre was identified. Supernatants from AFT024-hkirre could also support CD34+CD38? cells expansion. These results demonstrated that the AFT024-hKirre cells have the ability to efficiently expand HSCs/HPCs. PMID:24719861

  9. 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, 200?m 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. PMID:24575346

  10. Geometric modeling, functional parameter calculation, and visualization of the in-vivo distended rectal wall

    NASA Astrophysics Data System (ADS)

    Haider, Clifton R.; Manduca, Armando; Camp, Jon J.; Fletcher, Joel G.; Robb, Richard A.; Bharucha, Adil E.

    2006-03-01

    The rectum can distend to accommodate stool, and contracts in response to distention during defecation. Rectal motor dysfunctions are implicated in the pathophysiology of functional defecation disorders and fecal incontinence. These rectal motor functions can be studied by intra-luminal measurements of pressure by manometry, or combined with volume during rectal balloon distention. Pressure-volume (p-v) relationships provide a global index of rectal mechanical properties. However, balloon distention alone does not measure luminal radius or wall thickness, which are necessary to compute wall tension and stress respectively. It has been suggested that the elastic modulus, which is the linear slope of the stress-strain relationship, is a more accurate measure of wall stiffness. Also, measurements of compliance may not reflect differences in rectal diameter between subjects prior to inflation, and imaging is necessary to determine if, as has been suggested, rectal pressure-volume relationships are affected by extra-rectal structures. We have developed a technique to measure rectal stress:strain relationships in humans, by simultaneous magnetic resonance imaging (MRI) during rectal balloon distention. After a conditioning distention, a rectal balloon was distended with water from 0 to 400 ml in 50 ml steps, and imaged at each step with MRI. The fluid filled balloon was segmented from each volume, the phase-ordered binary volumes were transformed into a geometric characterization of the inflated rectal surface. Taken together with measurements of balloon pressure and of rectal wall thickness, this model of the rectal surface was used to calculate regional values of curvature, tension, strain, and stress for the rectum. In summary, this technique has the unique ability to non-invasively measure the rectal stress:strain relationship and also determine if rectal expansion is limited by extra-rectal structures. This functional information allows the direct clinical analysis of rectal motor function and offers the potential for characterizing abnormal mechanical properties of the rectal wall in disease.

  11. Evaluation of hybrid algorithm for analysis of scattered light using ex vivo nuclear morphology measurements of cervical epithelium.

    PubMed

    Ho, Derek; Drake, Tyler K; Bentley, Rex C; Valea, Fidel A; Wax, Adam

    2015-08-01

    We evaluate a new hybrid algorithm for determining nuclear morphology using angle-resolved low coherence interferometry (a/LCI) measurements in ex vivo cervical tissue. The algorithm combines Mie theory based and continuous wavelet transform inverse light scattering analysis. The hybrid algorithm was validated and compared to traditional Mie theory based analysis using an ex vivo tissue data set. The hybrid algorithm achieved 100% agreement with pathology in distinguishing dysplastic and non-dysplastic biopsy sites in the pilot study. Significantly, the new algorithm performed over four times faster than traditional Mie theory based analysis. PMID:26309741

  12. 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. PMID:26387954

  13. 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.

  14. Disruption of endocrine function in H295R cell in vitro and in zebrafish in vivo by naphthenic acids.

    PubMed

    Wang, Jie; Cao, Xiaofeng; Sun, Jinhua; Huang, Yi; Tang, Xiaoyan

    2015-12-15

    Oil sands process-affected water (OSPW) have been reported to exhibit endocrine disrupting effects on aquatic organisms. Although the responsible compounds are unknown, naphthenic acids (NAs) have been considered to be implicated. The current study was designed to investigate the endocrine disruption of OSPW extracted NAs (OS-NAs) and commercial NAs (C-NAs) using a combination of in vitro and in vivo assays. The effects of OS-NAs and C-NAs on steroidogenesis were assessed both at hormone levels and expression levels of hormone-related genes in the H295R cells. The transcriptions of biomarker genes involved in endocrine systems in zebrafish larvae were investigated to detect the effects of OS-NAs and C-NAs on endocrine function in vivo. Exposure to OS-NAs and C-NAs significantly increased production of 17?-estradiol (E2) and progesterone (P4), and decreased production of testosterone (T). Both OS-NAs and C-NAs significantly induced the expression of several genes involved in steroidogenesis. The abundances of transcripts of biomarker gene CYP19b, ER?, and VTG were significantly up-regulated in zebrafish larvae exposed to OS-NAs and C-NAs, which indicated that NAs had negative effects on estrogen-responsive gene transcription in vivo. These results indicated that NAs should be partly responsible for the endocrine disrupting effects of OSPW. PMID:26073515

  15. Fluorescein and radiolabeled Function-Spacer-Lipid constructs allow for simple in vitro and in vivo bioimaging of enveloped virions.

    PubMed

    Hadac, Elizabeth M; Federspiel, Mark J; Chernyy, Evgeny; Tuzikov, Alexander; Korchagina, Elena; Bovin, Nicolai V; Russell, Stephen; Henry, Stephen M

    2011-09-01

    Tools that can aid in vitro and in vivo imaging and also noninvasively determine half-life and biodistribution are required to advance clinical developments. A Function-Spacer-Lipid construct (FSL) incorporating fluorescein (FSL-FLRO4) was used to label vesicular stomatitis virus (VSV), measles virus MV-NIS (MV) and influenza virus (H1N1). The ability of FSL constructs to label these virions was established directly by FACScan of FSL-FLRO4 labeled VSV and MV, and indirectly following labeled H1N1 and MV binding to a cells. FSL-FLRO4 labeling of H1N1 was shown to maintain higher infectivity of the virus when compared with direct fluorescein virus labeling. A novel tyrosine (125)I radioiodinated FSL construct was synthesized (FSL-(125)I) from FSL-tyrosine. This was used to label VSV (VSV-FSL-(125)I), which was infused into the peritoneal cavity of laboratory mice. Bioscanning showed VSV-FSL-(125)I to localize in the liver, spleen and bloodstream in contrast to the free labels FSL-(125)I or (125)I, which localized predominantly in the liver and thyroid respectively. This is a proof-of-principle novel and rapid method for modifying virions and demonstrates the potential of FSL constructs to improve in vivo imaging of virions and noninvasively observe in vivo biodistribution. PMID:21703308

  16. Stochastic precision analysis of 2D cardiac strain estimation in vivo

    NASA Astrophysics Data System (ADS)

    Bunting, E. A.; Provost, J.; Konofagou, E. E.

    2014-11-01

    Ultrasonic strain imaging has been applied to echocardiography and carries great potential to be used as a tool in the clinical setting. Two-dimensional (2D) strain estimation may be useful when studying the heart due to the complex, 3D deformation of the cardiac tissue. Increasing the framerate used for motion estimation, i.e. motion estimation rate (MER), has been shown to improve the precision of the strain estimation, although maintaining the spatial resolution necessary to view the entire heart structure in a single heartbeat remains challenging at high MERs. Two previously developed methods, the temporally unequispaced acquisition sequence (TUAS) and the diverging beam sequence (DBS), have been used in the past to successfully estimate in vivo axial strain at high MERs without compromising spatial resolution. In this study, a stochastic assessment of 2D strain estimation precision is performed in vivo for both sequences at varying MERs (65, 272, 544, 815?Hz for TUAS; 250, 500, 1000, 2000?Hz for DBS). 2D incremental strains were estimated during left ventricular contraction in five healthy volunteers using a normalized cross-correlation function and a least-squares strain estimator. Both sequences were shown capable of estimating 2D incremental strains in vivo. The conditional expected value of the elastographic signal-to-noise ratio (E(SNRe|?)) was used to compare strain estimation precision of both sequences at multiple MERs over a wide range of clinical strain values. The results here indicate that axial strain estimation precision is much more dependent on MER than lateral strain estimation, while lateral estimation is more affected by strain magnitude. MER should be increased at least above 544?Hz to avoid suboptimal axial strain estimation. Radial and circumferential strain estimations were influenced by the axial and lateral strain in different ways. Furthermore, the TUAS and DBS were found to be of comparable precision at similar MERs.

  17. 20-HETE Regulates the Angiogenic Functions of Human Endothelial Progenitor Cells and Contributes to Angiogenesis In Vivo

    PubMed Central

    Chen, Li; Ackerman, Rachel; Saleh, Mohamed; Gotlinger, Katherine H.; Kessler, Michael; Mendelowitz, Lawrence G.; Falck, John R.; Arbab, Ali S.; Scicli, A. Guillermo; Schwartzman, Michal L.

    2014-01-01

    Circulating endothelial progenitor cells (EPC) contribute to postnatal neovascularization. We identified the cytochrome P450 4A/F–20-hydroxyeicosatetraenoic acid (CYP4A/F–20-HETE) system as a novel regulator of EPC functions associated with angiogenesis in vitro. Here, we explored cellular mechanisms by which 20-HETE regulates EPC angiogenic functions and assessed its contribution to EPC-mediated angiogenesis in vivo. Results showed that both hypoxia and vascular endothelial growth factor (VEGF) induce CYP4A11 gene and protein expression (the predominant 20-HETE synthases in human EPC), and this is accompanied by an increase in 20-HETE production by ?1.4- and 1.8-fold, respectively, compared with the control levels. Additional studies demonstrated that 20-HETE and VEGF have a synergistic effect on EPC proliferation, whereas 20-HETE antagonist 20-HEDGE or VEGF-neutralizing antibody negated 20-HETE- or VEGF-induced proliferation, respectively. These findings are consistent with the presence of a positive feedback regulation on EPC proliferation between the 20-HETE and the VEGF pathways. Furthermore, we found that 20-HETE induced EPC adhesion to fibronectin and endothelial cell monolayer by 40 ± 5.6 and 67 ± 10%, respectively, which was accompanied by a rapid induction of very late antigen-4 and chemokine receptor type 4 mRNA and protein expression. Basal and 20-HETE-stimulated increases in adhesion were negated by the inhibition of the CYP4A–20-HETE system. Lastly, EPC increased angiogenesis in vivo by 3.6 ± 0.2-fold using the Matrigel plug angiogenesis assay, and these increases were markedly reduced by the local inhibition of 20-HETE system. These results strengthened the notion that 20-HETE regulates the angiogenic functions of EPC in vitro and EPC-mediated angiogenesis in vivo. PMID:24403517

  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. PMID:25693608

  19. In Vivo Assessment of Endothelial Function in Human Lower Extremity Arteries

    PubMed Central

    Kashyap, Vikram S.; Lakin, Ryan O.; Feiten, Lindsay E.; Bishop, Paul; Sarac, Timur P.

    2013-01-01

    Objective Endothelial function has been measured in preclinical studies, in human brachial and coronary arteries, but not in lower extremity arteries affected by atherosclerosis. We describe a novel, first-in-man, evaluation of endothelial function of the superficial femoral arteries (SFA) in patients with peripheral arterial disease (PAD). Methods Patients with PAD (n=25) requiring lower extremity angiography were enrolled. Endothelial dependent relaxation (EDR) was measured using intravascular ultrasound and a Doppler Flow wire after the infusion of acetylcholine (Ach). IVUS derived virtual histology (IVUS-VH) of the same vessel was calculated. Endothelial independent relaxation (EIR) was measured with infusion of nitroglycerin (NTG, 200 µg). Levels of nitric oxide (NOx) and serum metabolites were determined by laboratory analysis. Results Patients (mean age 62, 48% male) had a history of hypertension (80%), coronary disease (36%), and diabetes (40%). The mean SFA diameter was 5.2 ± 1 mm (range 3.2–6.9 mm). Patients tolerated Ach infusion with no side effects or adverse events. EDR increased over baseline for all patients with Ach infusion 10?6-10?4. Diameter (0.5% at Ach 10?4) and area (1.8% at Ach 10?4) changes in the diseased SFA were modest and insignificant. But, average peak velocity of blood flow (APV) significantly increased 26, 46 and 63% with Ach infusion 10?6-10?4. Calculations of limb volumetric flow (Q, mL/s, 68%, Ach 10?4) were significantly increased after Ach infusion. Lower extremity NOx levels were slightly lower than systemic venous levels (P = .04). NTG infusion indicated normal smooth muscle responsiveness (3% diameter, 9% area, and 116% velocity change over baseline). IVUS-VH plaque stratification indicated predominantly fibrous morphology (46%; necrotic core, 29%; calcium, 18%). Atheroma burden was 14.9 ± 5.5 mm3/cm and did not correlate with endothelial responsiveness. Conclusions Endothelial function can be measured directly in human lower extremity arteries at the sites of vascular disease. Despite extensive atherosclerosis, endothelial function is still intact. These data support the application of regional endothelial-specific biological therapies in patients with PAD. PMID:23830159

  20. Arginyltransferase is an ATP-Independent Self-Regulating Enzyme that Forms Distinct Functional Complexes In Vivo

    PubMed Central

    Wang, Junling; Han, Xuemei; Saha, Sougata; Xu, Tao; Rai, Reena; Zhang, Fangliang; Wolf, Yuri. I.; Wolfson, Alexey; Yates, John R.; Kashina, Anna

    2010-01-01

    Summary Posttranslational arginylation mediated by arginyltransferase (ATE1) plays an important role in cardiovascular development, cell motility and regulation of cytoskeleton and metabolic enzymes. This protein modification was discovered decades ago, however, the arginylation reaction and the functioning of ATE1 remained poorly understood due to the lack of good biochemical models. Here we report the development of an in vitro arginylation system, in which ATE1 function and molecular requirements can be tested using purified recombinant ATE1 isoforms supplemented with a controlled number of components. Our results show that arginylation reaction is a self-sufficient, ATP-independent process that can affect different sites in a polypeptide, and that arginyltransferases form different molecular complexes in vivo, associate with components of the translation machinery, and have distinct, partially overlapping subsets of substrates, suggesting that these enzymes play different physiological functions. PMID:21276945

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

    PubMed

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

    2015-07-13

    The use of click chemistry reactions for the functionalization of nanoparticles is particularly useful to modify the surface in a well-defined manner and to enhance the targeting properties, thus facilitating clinical translation. Here it is demonstrated that olefin metathesis can be used for the chemoselective functionalization of iron oxide nanoparticles with three different examples. This approach enables, in one step, the synthesis and functionalization of different water-stable magnetite-based particles from oleic acid-coated counterparts. The surface of the nanoparticles was completely characterized showing how the metathesis approach introduces a large number of hydrophilic molecules on their coating layer. As an example of the possible applications of these new nanocomposites, a focus was taken on atherosclerosis plaques. It is also demonstrated how the in vitro properties of one of the probes, particularly its Ca(2+) -binding properties, mediate their final in vivo use; that is, the selective accumulation in atherosclerotic plaques. This opens promising new applications to detect possible microcalcifications associated with plaque vulnerability. The accumulation of the new imaging tracers is demonstrated by in vivo magnetic resonance imaging of carotids and aorta in the ApoE(-/-) mouse model and the results were confirmed by histology. PMID:26096657

  2. 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

  3. Functional expression of low density lipoprotein receptor-related protein is controlled by receptor-associated protein in vivo.

    PubMed Central

    Willnow, T E; Armstrong, S A; Hammer, R E; Herz, J

    1995-01-01

    The 39-kDa receptor-associated protein (RAP) associates with the multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) and thereby prevents the binding of all known ligands, including alpha 2-macroglobulin and chylomicron remnants. RAP is predominantly localized in the endoplasmic reticulum, raising the possibility that it functions as a chaperone or escort protein in the biosynthesis or intracellular transport of LRP. Here we have used gene targeting to show that RAP promotes the expression of functional LRP in vivo. The amount of mature, processed LRP is reduced in liver and brain of RAP-deficient mice. As a result, hepatic clearance of alpha 2-macroglobulin is impaired and remnant lipoproteins accumulate in the plasma of RAP-deficient mice that also lack functional LDL receptors. These results are consistent with the hypothesis that RAP stabilizes LRP within the secretory pathway. They also suggest a further mechanism by which the activity of an endocytic receptor may be modulated in vivo. Images Fig. 2 Fig. 5 PMID:7538675

  4. Integrity of prokaryotic mRNA isolated from complex samples for in vivo bacterial transcriptome analysis.

    PubMed

    Ferreira-Machado, A B; Freitas, M C R; Saji, G R Q; Rezende, A B; Almeida, P E; Cesar, D E; Resende, J A; Nicólas, M F; Silva, V L; Diniz, C G

    2015-01-01

    Even though several in vitro studies have focused on bacterial biology, the extent of such knowledge is not complete when considering an actual infection. As culture-independent microbiology methods such as high-throughput sequencing became available, important aspects of host-bacterium interactions will be elucidated. Based on microbiological relevance, we considered Bacteroides fragilis in a murine experimental infection as a model system to evaluate the in vivo bacterial transcriptome in host exudates. A disproportionate number of reads belonging to the host genome were retrieved in the first round of pyrosequencing, even after depletion of ribosomal RNA; the average number of reads related to the eukaryotic genome was 71.924-67.7%, whereas prokaryotic reads represented 34.338-32.3% in host exudates. Thus, different treatments were used to improve the prokaryotic RNA yield: i) centrifugation; ii) ultrasonic treatment; and iii) ultrasonic treatment followed by centrifugation. The latter treatment was found to be the most efficient in generating bacterial yields, as it resulted in a higher number of Bacteroides cells. However, the RNA extracted after this treatment was not of sufficient quality to be used in cDNA synthesis. Our results suggest that the methodology routinely used for RNA extraction in transcriptional analysis is not appropriate for in vivo studies in complex samples. Furthermore, the most efficient treatment for generating good bacterial cell yields was not suitable to retrieve high-quality RNA. Therefore, as an alternative methodological approach to enable in vivo studies on host-bacterium interactions, we advise increasing the sequencing depth despite the high costs. PMID:26600536

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

    E-print Network

    Yaung, Stephanie J.

    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 ...

  6. Genome-wide analysis of ETS-family DNA-binding in vitro and in vivo

    PubMed Central

    Wei, Gong-Hong; Badis, Gwenael; Berger, Michael F; Kivioja, Teemu; Palin, Kimmo; Enge, Martin; Bonke, Martin; Jolma, Arttu; Varjosalo, Markku; Gehrke, Andrew R; Yan, Jian; Talukder, Shaheynoor; Turunen, Mikko; Taipale, Mikko; Stunnenberg, Hendrik G; Ukkonen, Esko; Hughes, Timothy R; Bulyk, Martha L; Taipale, Jussi

    2010-01-01

    Members of the large ETS family of transcription factors (TFs) have highly similar DNA-binding domains (DBDs)—yet they have diverse functions and activities in physiology and oncogenesis. Some differences in DNA-binding preferences within this family have been described, but they have not been analysed systematically, and their contributions to targeting remain largely uncharacterized. We report here the DNA-binding profiles for all human and mouse ETS factors, which we generated using two different methods: a high-throughput microwell-based TF DNA-binding specificity assay, and protein-binding microarrays (PBMs). Both approaches reveal that the ETS-binding profiles cluster into four distinct classes, and that all ETS factors linked to cancer, ERG, ETV1, ETV4 and FLI1, fall into just one of these classes. We identify amino-acid residues that are critical for the differences in specificity between all the classes, and confirm the specificities in vivo using chromatin immunoprecipitation followed by sequencing (ChIP-seq) for a member of each class. The results indicate that even relatively small differences in in vitro binding specificity of a TF contribute to site selectivity in vivo. PMID:20517297

  7. In vivo analysis of highly conserved Nef activities in HIV-1 replication and pathogenesis

    PubMed Central

    2013-01-01

    Background The HIV-1 accessory protein, Nef, is decisive for progression to AIDS. In vitro characterization of the protein has described many Nef activities of unknown in vivo significance including CD4 downregulation and a number of activities that depend on Nef interacting with host SH3 domain proteins. Here, we use the BLT humanized mouse model of HIV-1 infection to assess their impact on viral replication and pathogenesis and the selection pressure to restore these activities using enforced in vivo evolution. Results We followed the evolution of HIV-1LAI (LAI) with a frame-shifted nef (LAINeffs) during infection of BLT mice. LAINeffs was rapidly replaced in blood by virus with short deletions in nef that restored the open reading frame (LAINeffs?-1 and LAINeffs?-13). Subsequently, LAINeffs?-1 was often replaced by wild type LAI. Unexpectedly, LAINeffs?-1 and LAINeffs?-13 Nefs were specifically defective for CD4 downregulation activity. Viruses with these mutant nefs were used to infect BLT mice. LAINeffs?-1 and LAINeffs?-13 exhibited three-fold reduced viral replication (compared to LAI) and a 50% reduction of systemic CD4+ T cells (>90% for LAI) demonstrating the importance of CD4 downregulation. These results also demonstrate that functions other than CD4 downregulation enhanced viral replication and pathogenesis of LAINeffs?-1 and LAINeffs?-13 compared to LAINeffs. To gain insight into the nature of these activities, we constructed the double mutant P72A/P75A. Multiple Nef activities can be negated by mutating the SH3 domain binding site (P72Q73V74P75L76R77) to P72A/P75A and this mutation does not affect CD4 downregulation. Virus with nef mutated to P72A/P75A closely resembled the wild-type virus in vivo as viral replication and pathogenesis was not significantly altered. Unlike LAINeffs described above, the P72A/P75A mutation had a very weak tendency to revert to wild type sequence. Conclusions The in vivo phenotype of Nef is significantly dependent on CD4 downregulation but minimally on the numerous Nef activities that require an intact SH3 domain binding motif. These results suggest that CD4 downregulation plus one or more unknown Nef activities contribute to enhanced viral replication and pathogenesis and are suitable targets for anti-HIV therapy. Enforced evolution studies in BLT mice will greatly facilitate identification of these critical activities. PMID:24172637

  8. A novel RNA oligonucleotide improves liver function and inhibits liver carcinogenesis in vivo

    PubMed Central

    Reebye, V.; Sćtrom, P.; Mintz, P.J.; Huang, K.W.; Swiderski, P.; Peng, L.; Liu, C.; Liu, X.X.; Jensen, S.; Zacharoulis, D.; Kostomitsopoulos, N.; Kasahara, N.; Nicholls, J.P.; Jiao, L.R.; Pai, M.; Mizandari, M.; Chikovani, T.; Emara, M.M.; Haoudi, A.; Tomalia, D.A.; Rossi, J.J.; Habib, N.A.; Spalding, D.R.

    2015-01-01

    Hepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here, we show an innovative RNA-based targeted approach to enhance endogenous albumin production whilst reducing liver tumour burden. We designed short-activating RNAs (saRNA) to enhance expression of C/EBP? (CCAAT/enhancer-binding protein-?), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBP? and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBP?-saRNA transfected HepG2 cells. Intravenous injection of C/EBP?-saRNA in a cirrhotic rat model with multifocal liver tumours increased circulating serum albumin by over 30% showing evidence of improved liver function. Tumour burden decreased by 80% (p = 0.003) with a 40% reduction in a marker of pre-neoplastic transformation. Since C/EBP? has known anti-proliferative activities via retinoblastoma, p21 and cyclins; we used mRNA expression liver cancer specific microarray in C/EBP?-saRNA transfected HepG2 cells to confirm down-regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis and metastasis. Up-regulated genes were enriched for tumour suppressors and positive regulators of cell differentiation. A quantitative PCR and Western-blot analysis of C/EBP?-saRNA transfected cells suggested that in addition to the known anti-proliferative targets of C/EBP?, we also observed suppression of IL6R, c-Myc and reduced STAT3 phosphorylation. Conclusion We demonstrate for the first time that a novel injectable saRNA-oligonucleotide that enhances C/EBP? expression successfully reduces tumour burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model. PMID:23929703

  9. 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.

  10. 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.

  11. 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 ?m) 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 ?m. 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.

  12. In vivo evolution of metabolic pathways: Assembling old parts to build novel and functional structures

    PubMed Central

    Luque, Alejandro; Sebai, Sarra C; Sauveplane, Vincent; Ramaen, Odile; Pandjaitan, Rudy

    2014-01-01

    In our recent article “In vivo evolution of metabolic pathways by homeologous recombination in mitotic cells” we proposed a useful alternative to directed evolution methods that permits the generation of yeast cell libraries containing recombinant metabolic pathways from counterpart genes. The methodology was applied to generate single mosaic genes and intragenic mosaic pathways. We used flavonoid metabolism genes as a working model to assembly and express evolved pathways in DNA repair deficient cells. The present commentary revises the principles of gene and pathway mosaicism and explores the scope and perspectives of our results as an additional tool for synthetic biology. PMID:25482082

  13. 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

  14. Generation and characterization of androgen receptor knockout (ARKO) mice: An in vivo model for the study of androgen functions in selective tissues

    PubMed Central

    Yeh, Shuyuan; Tsai, Meng-Yin; Xu, Qingquan; Mu, Xiao-Min; Lardy, Henry; Huang, Ko-En; Lin, Hank; Yeh, Shauh-Der; Altuwaijri, Saleh; Zhou, Xinchang; Xing, Lianping; Boyce, Brendan F.; Hung, Min-Chi; Zhang, Su; Gan, Lin; Chang, Chawnshang

    2002-01-01

    By using a cre-lox conditional knockout strategy, we report here the generation of androgen receptor knockout (ARKO) mice. Phenotype analysis shows that ARKO male mice have a female-like appearance and body weight. Their testes are 80% smaller and serum testosterone concentrations are lower than in wild-type (wt) mice. Spermatogenesis is arrested at pachytene spermatocytes. The number and size of adipocytes are also different between the wt and ARKO mice. Cancellous bone volumes of ARKO male mice are reduced compared with wt littermates. In addition, we found the average number of pups per litter in homologous and heterozygous ARKO female mice is lower than in wt female mice, suggesting potential defects in female fertility and/or ovulation. The cre-lox ARKO mouse provides a much-needed in vivo animal model to study androgen functions in the selective androgen target tissues in female or male mice. PMID:12370412

  15. Enhancer Analysis Unveils Genetic Interactions between TLX and SOX2 in Neural Stem Cells and In Vivo Reprogramming

    PubMed Central

    Islam, Mohammed M.; Smith, Derek K.; Niu, Wenze; Fang, Sanhua; Iqbal, Nida; Sun, Guoqiang; Shi, Yanhong; Zhang, Chun-Li

    2015-01-01

    Summary The orphan nuclear receptor TLX is a master regulator of postnatal neural stem cell (NSC) self-renewal and neurogenesis; however, it remains unclear how TLX expression is precisely regulated in these tissue-specific stem cells. Here, we show that a highly conserved cis-element within the Tlx locus functions to drive gene expression in NSCs. We demonstrate that the transcription factors SOX2 and MYT1 specifically interact with this genomic element to directly regulate Tlx enhancer activity in vivo. Knockdown experiments further reveal that SOX2 dominantly controls endogenous expression of TLX, whereas MYT1 only plays a modulatory role. Importantly, TLX is essential for SOX2-mediated in vivo reprogramming of astrocytes and itself is also sufficient to induce neurogenesis in the adult striatum. Together, these findings unveil functional genetic interactions among transcription factors that are critical to NSCs and in vivo cell reprogramming. PMID:26607952

  16. Structural and Functional Analysis of Saccharomyces Cerevisiae Mob1

    SciTech Connect

    Mrkobrada,S.; Boucher, L.; Tyers, D.; Sicheri, F.

    2006-01-01

    The Mob proteins function as activator subunits for the Dbf2/Dbf20 family of protein kinases. Human and Xenopus Mob1 protein structures corresponding to the most conserved C-terminal core, but lacking the variable N-terminal region, have been reported and provide a framework for understanding the mechanism of Dbf2/Dbf20 regulation. Here, we report the 2.0 {angstrom} X-ray crystal structure of Saccharomyces cerevisiae Mob1 containing both the conserved C-terminal core and the variable N-terminal region. Within the N-terminal region, three novel structural elements are observed; namely, an {alpha}-helix denoted H0, a strand-like element denoted S0 and a short {beta} strand denoted S-1. Helix H0 associates in an intermolecular manner with a second Mob1 molecule to form a Mob1 homodimer. Strand S0 binds to the core domain in an intramolecular manner across a putative Dbf2 binding site mapped by Mob1 temperature-sensitive alleles and NMR binding experiments. In vivo functional analysis demonstrates that Mob1 mutants that target helix H0 or its reciprocal binding site are biologically compromised. The N-terminal region of Mob1 thus contains structural elements that are functionally important.

  17. Impact of nonnatural amino acid mutagenesis on the in vivo function and binding modes of a transcriptional activator.

    PubMed

    Majmudar, Chinmay Y; Lee, Lori W; Lancia, Jody K; Nwokoye, Adaora; Wang, Qian; Wands, Amberlyn M; Wang, Lei; Mapp, Anna K

    2009-10-14

    Protein-protein interactions play an essential role in cellular function, and methods to discover and characterize them in their native context are of paramount importance for gaining a deeper understanding of biological networks. In this study, an enhanced nonsense suppression system was utilized to incorporate the nonnatural amino acid p-benzoyl-L-phenylalanine (pBpa) throughout the transcriptional activation domain of the prototypical eukaryotic transcriptional activator Gal4 in vivo (S. cerevisiae). Functional studies of the pBpa-containing Gal4 mutants suggest that this essential binding interface of Gal4 is minimally impacted by these substitutions, with both transcriptional activity and sensitivity to growth conditions maintained. Further supporting this are in vivo cross-linking studies, including the detection of a key binding partner of Gal4, the inhibitor protein Gal80. Cross-linking with a range of pBpa-containing mutants revealed a Gal4 x Gal80 binding interface that extends beyond that previously predicted by conventional strategies. Thus, this approach can be broadened to the discovery of novel binding partners of transcription factors, information that will be critical for the development of therapeutically useful small molecule modulators of these protein-protein interactions. PMID:19764747

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

    PubMed Central

    Pagčs, Stéphane; Cane, Michele; Randall, Jérôme; Capello, Luca; Holtmaat, Anthony

    2015-01-01

    Longitudinal imaging studies of neuronal structures in vivo have revealed rich dynamics in dendritic spines and axonal boutons. Spines and boutons are considered to be proxies for synapses. This implies that synapses display similar dynamics. However, spines and boutons do not always bear synapses, some may contain more than one, and dendritic shaft synapses have no clear structural proxies. In addition, synaptic strength is not always accurately revealed by just the size of these structures. Structural and functional dynamics of synapses could be studied more reliably using fluorescent synaptic proteins as markers for size and function. These proteins are often large and possibly interfere with circuit development, which renders them less suitable for conventional transfection or transgenesis methods such as viral vectors, in utero electroporation, and germline transgenesis. Single cell electroporation (SCE) has been shown to be a potential alternative for transfection of recombinant fluorescent proteins in adult cortical neurons. Here we provide proof of principle for the use of SCE to express and subsequently image fluorescently tagged synaptic proteins over days to weeks in vivo. PMID:25904849

  19. Biocompatible near-infrared fluorescent nanoparticles for macro and microscopic in vivo functional bioimaging

    PubMed Central

    Chu, Liliang; Wang, Shaowei; Li, Kanghui; Xi, Wang; Zhao, Xinyuan; Qian, Jun

    2014-01-01

    Near-infrared (NIR) imaging technology has been widely used for biomedical research and applications, since it can achieve deep penetration in biological tissues due to less absorption and scattering of NIR light. In our research, polymer nanoparticles with NIR fluorophores doped were synthesized. The morphology, absorption/emission features and chemical stability of the fluorescent nanoparticles were characterized, separately. NIR fluorescent nanoparticles were then utilized as bright optical probes for macro in vivo imaging of mice, including sentinel lymph node (SLN) mapping, as well as distribution and excretion monitoring of nanoparticles in animal body. Furthermore, we applied the NIR fluorescent nanoparticles in in vivo microscopic bioimaging via a confocal microscope. Under the 635 nm-CW excitation, the blood vessel architecture in the ear and the brain of mice, which were administered with nanoparticles, was visualized very clearly. The imaging depth of our one-photon microscopy, which was assisted with NIR fluorescent nanoprobes, can reach as deep as 500 ?m. Our experiments show that NIR fluorescent nanoparticles have great potentials in various deep-tissue imaging applications. PMID:25426331

  20. In Vitro and In Vivo Tumor Targeted Photothermal Cancer Therapy Using Functionalized Graphene Nanoparticles.

    PubMed

    Kim, Sung Han; Lee, Jung Eun; Sharker, Shazid Md; Jeong, Ji Hoon; In, Insik; Park, Sung Young

    2015-11-01

    Despite the tremendous progress that photothermal therapy (PTT) has recently achieved, it still has a long way to go to gain the effective targeted photothermal ablation of tumor cells. Driven by this need, we describe a new class of targeted photothermal therapeutic agents for cancer cells with pH responsive bioimaging using near-infrared dye (NIR) IR825, conjugated poly(ethylene glycol)-g-poly(dimethylaminoethyl methacrylate) (PEG-g-PDMA, PgP), and hyaluronic acid (HA) anchored reduced graphene oxide (rGO) hybrid nanoparticles. The obtained rGO nanoparticles (PgP/HA-rGO) showed pH-dependent fluorescence emission and excellent near-infrared (NIR) irradiation of cancer cells targeted in vitro to provide cytotoxicity. Using intravenously administered PTT agents, the time-dependent in vivo tumor target accumulation was exactly defined, presenting eminent photothermal conversion at 4 and 8 h post-injection, which was demonstrated from the ex vivo biodistribution of tumors. These tumor environment responsive hybrid nanoparticles generated photothermal heat, which caused dominant suppression of tumor growth. The histopathological studies obtained by H&E staining demonstrated complete healing from malignant tumor. In an area of limited successes in cancer therapy, our translation will pave the road to design stimulus environment responsive targeted PTT agents for the safe eradication of devastating cancer. PMID:26451914

  1. Chromatin-modifying agents promote the ex vivo production of functional human erythroid progenitor cells.

    PubMed

    Chaurasia, Pratima; Berenzon, Dmitriy; Hoffman, Ronald

    2011-04-28

    Presently, blood transfusion products (TPs) are composed of terminally differentiated cells with a finite life span. We have developed an ex vivo-generated TP composed of erythroid progenitor cells (EPCs) and precursors cells. Several histone deacetylase inhibitors (HDACIs) were used in vitro to promote the preferential differentiation of cord blood (CB) CD34(+) cells to EPCs. A combination of cytokines and valproic acid (VPA): (1) promoted the greatest degree of EPC expansion, (2) led to the generation of EPCs which were capable of differentiating into the various stages of erythroid development, (3) led to epigenetic modifications (increased H3 acetylation) of promoters for erythroid-specific genes, which resulted in the acquisition of a gene expression pattern characteristic of primitive erythroid cells, and (4) promoted the generation of a TP that when infused into NOD/SCID mice produced mature RBCs containing both human adult and fetal globins as well Rh blood group Ag which persisted for 3 weeks and the retention of human EPCs and erythroid precursor cells within the BM of recipient mice. This ex vivo-generated EPC-TP likely represents a paradigm shift in transfusion medicine because of its potential to continue to generate additional RBCs after its infusion. PMID:21355088

  2. 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. Environ. Mol. Mutagen. 56:788-793, 2015. © 2015 Wiley Periodicals, Inc. PMID:26248301

  3. 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.

  4. The relationship between the diameter of chemically-functionalized multi-walled carbon nanotubes and their organ biodistribution profiles in vivo.

    PubMed

    Wang, Julie T-W; Fabbro, Chiara; Venturelli, Enrica; Ménard-Moyon, Cécilia; Chaloin, Olivier; Da Ros, Tatiana; Methven, Laura; Nunes, Antonio; Sosabowski, Jane K; Mather, Stephen J; Robinson, Martyn K; Amadou, Julien; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas; Al-Jamal, Khuloud T

    2014-11-01

    Carbon nanotubes (CNTs) exhibit unique properties which have led to their applications in the biomedical field as novel delivery systems for diagnosis and therapy purposes. We have previously reported that the degree of functionalization of CNTs is a key factor determining their biological behaviour. The present study broadens the spectrum by investigating the impact of the diameter of CNTs using two series of multi-walled CNTs (MWNTs) with distinct differences in their diameters. Both MWNTs were doubly functionalized by 1,3-dipolar cycloaddition and amidation reactions, allowing the appended functional groups to be further conjugated with radionuclide chelating moieties and antibodies or antibody fragments. All constructs possessed comparable degree of functionalization and were characterized by thermogravimetric analysis, transmission electron microscopy, gel electrophoresis and surface plasmon resonance. The MWNT conjugates were radio-labelled with indium-111, which thereby enabled in vivo single photon emission computed tomography/computed tomography (SPECT/CT) imaging and organ biodistribution study using ?-scintigraphy. The narrow MWNTs (average diameter: 9.2 nm) demonstrated enhanced tissue affinity including non-reticular endothelial tissues compared to the wider MWNTs (average diameter: 39.5 nm). The results indicate that the higher aspect ratio of narrow MWNTs may be beneficial for their future biological applications due to higher tissue accumulation. PMID:25168822

  5. In Vivo Readout of CFTR Function: Ratiometric Measurement of CFTR-Dependent Secretion by Individual, Identifiable Human Sweat Glands

    PubMed Central

    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 ?-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. PMID:24204751

  6. GPU accelerated dynamic functional connectivity analysis for functional MRI data.

    PubMed

    Akgün, Devrim; Sako?lu, 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. PMID:25805449

  7. Cyclosporine augments renal mitochondrial function in vivo and reduces renal blood flow.

    PubMed

    Lemmi, C A; Pelikan, P C; Sikka, S C; Hirschberg, R; Geesaman, B; Miller, R L; Park, K S; Liu, S C; Koyle, M; Rajfer, J

    1989-11-01

    The in vivo action of cyclosporine A (CS) on rat renal cortical mitochondria was investigated. CS (30 mg.kg-1.day-1) given orally to rats for 30 days caused an augmentation of renal mitochondrial oxidative phosphorylation. The ADP-stimulated respiratory rate was increased by 37.0% with glutamate plus malate as respiratory substrates (P less than 0.025) but not with succinate-supported respiration, indicating enhancement of mitochondrial complex I activity. This reaction may be a response to the 32.5% reduction of renal blood (P less than 0.005) in the CS-treated group, possibly serving to maximize ATP synthesis during ischemia. Ligation-induced decreases in renal blood flow also resulted in enhancement of mitochondrial complex I activity. PMID:2589485

  8. 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. PMID:25783412

  9. 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.

  10. In vivo transcriptional profile analysis reveals RNA splicing and chromatin remodeling as prominent processes for adult neurogenesis

    E-print Network

    In vivo transcriptional profile analysis reveals RNA splicing and chromatin remodeling as prominent of data from these three separate approaches showed that in adult SVZ neurogenesis, RNA splicing, transcription, and neurogenesis. In non-neurogenic brain regions, RNA splicing and chromatin remodeling were

  11. The C2 domain of phosphatidylserine decarboxylase 2 is not required for catalysis but is essential for in vivo function.

    PubMed

    Kitamura, Hidemitsu; Wu, Wen-I; Voelker, Dennis R

    2002-09-13

    Phosphatidylserine decarboxylase 2 (Psd2p) is currently being used to study lipid trafficking processes in intact and permeabilized yeast cells. The Psd2p contains a C2 homology domain and a putative Golgi retention/localization (GR) domain. C2 domains play important functions in membrane binding and docking reactions involving phospholipids and proteins. We constructed a C2 domain deletion variant (C2Delta) and a GR deletion variant (GRDelta) of Psd2p and examined their effects on in vivo function and catalysis. Immunoblotting confirmed that the predicted immature and mature forms of Psd2(C2Delta)p, Psd2(GRDelta)p, and wild type Psd2p were produced in vivo and that the proteins localized normally. Enzymology revealed that the Psd2(C2Delta)p and Psd2(GRDelta)p were catalytically active and could readily be expressed at levels 10-fold higher than endogenous Psd2p. Both Psd2p and Psd2(GRDelta)p expression complemented the growth defect of psd1Deltapsd2Delta strains and resulted in normal aminoglycerophospholipid metabolism. In contrast, the Psd2(C2Delta)p failed to complement psd1Deltapsd2Delta strains, and [(3)H]serine labeling revealed a severe defect in the formation of PtdEtn in both intact and permeabilized cells, indicative of disruption of lipid trafficking. These findings identify an essential, non-catalytic function of the C2 domain of Psd2p and raise the possibility that it plays a direct role in membrane docking and/or PtdSer transport to the enzyme. PMID:12093819

  12. Microarray analysis in rat liver slices correctly predicts in vivo hepatotoxicity

    SciTech Connect

    Elferink, M.G.L. Olinga, P.; Draaisma, A.L.; Merema, M.T.; Bauerschmidt, S.; Polman, J.; Schoonen, W.G.; Groothuis, G.M.M.

    2008-06-15

    The microarray technology, developed for the simultaneous analysis of a large number of genes, may be useful for the detection of toxicity in an early stage of the development of new drugs. The effect of different hepatotoxins was analyzed at the gene expression level in the rat liver both in vivo and in vitro. As in vitro model system the precision-cut liver slice model was used, in which all liver cell types are present in their natural architecture. This is important since drug-induced toxicity often is a multi-cellular process involving not only hepatocytes but also other cell types such as Kupffer and stellate cells. As model toxic compounds lipopolysaccharide (LPS, inducing inflammation), paracetamol (necrosis), carbon tetrachloride (CCl{sub 4}, fibrosis and necrosis) and gliotoxin (apoptosis) were used. The aim of this study was to validate the rat liver slice system as in vitro model system for drug-induced toxicity studies. The results of the microarray studies show that the in vitro profiles of gene expression cluster per compound and incubation time, and when analyzed in a commercial gene expression database, can predict the toxicity and pathology observed in vivo. Each toxic compound induces a specific pattern of gene expression changes. In addition, some common genes were up- or down-regulated with all toxic compounds. These data show that the rat liver slice system can be an appropriate tool for the prediction of multi-cellular liver toxicity. The same experiments and analyses are currently performed for the prediction of human specific toxicity using human liver slices.

  13. Inverse computational analysis of in vivo corneal elastic modulus change after collagen crosslinking for keratoconus

    PubMed Central

    Sinha Roy, Abhijit; Rocha, Karol M.; Randleman, J. Bradley; Stulting, R. Doyle; Dupps, William J.

    2014-01-01

    Corneal collagen crosslinking with riboflavin photosensitization and ultraviolet irradiation is a novel approach to limiting the progression of keratoconus in patients by increasing the elastic modulus of the degenerate cornea. Beneficial reductions in corneal steepness and aberrations after crosslinking also frequently occur. In a previous study, we described a computational modeling approach to simulating topographic progression in keratoconus and regression of disease with corneal collagen crosslinking. In the current study, this model has been expanded and applied to the inverse problem of estimating longitudinal time-dependent changes in the corneal elastic modulus after crosslinking using in vivo measurements from 16 human eyes. Topography measured before crosslinking was used to construct a patient-specific finite element model with assumed hyperelastic properties. Then the properties of the cornea were altered using an inverse optimization method to minimize the difference between the model-predicted and in vivo corneal shape after crosslinking. Effects of assumptions regarding sclera-to-cornea elastic modulus ratio and spatial attenuation of treatment effect due to ultraviolet beam characteristics on the predicted change in elastic modulus were also investigated. Corneal property changes computed by inverse finite element analysis provided excellent geometric agreement with clinical topography measurements in patient eyes post-crosslinking. Over all post-treatment time points, the estimated increase in corneal elastic modulus was 110.8±48.1%, and slightly less stiffening was required to produce the same amount of corneal topographic regression of disease when the sclera-to-cornea modulus ratio was increased. Including the effect of beam attenuation resulted in greater estimates of stiffening in the anterior cornea. Corneal shape responses to crosslinking varied considerably and emphasize the importance of a patient-specific approach. PMID:23664859

  14. A preclinical model for the analysis of genetically modified human skin in vivo.

    PubMed

    Del Rio, Marcela; Larcher, Fernando; Serrano, Fernando; Meana, Alvaro; Muńoz, Marta; Garcia, Marta; Muńoz, Evangelina; Martin, Clara; Bernad, Antonio; Jorcano, José Luis

    2002-05-20

    Although skin is perhaps the most accessible of all somatic tissues for therapeutic gene transfer, it is a challenging site when attempting gene delivery. In addition to the transience of gene expression, important obstacles to cutaneous gene therapy have included the inability to sustain gene expression in a large proportion of keratinocytes within a given skin compartment. In this study, we have developed a novel experimental strategy that allows long-term regeneration of entirely genetically engineered human skin on the backs of NOD/SCID mice. Primary human keratinocytes were infected with a retroviral vector encoding the enhanced green fluorescent protein (EGFP) produced by transient transfection of 293T cells. EGFP expression allowed cell-sorting selection of a polyclonal population of productively transduced keratinocytes that were assembled in a live fibroblast-containing fibrin dermal matrix and orthotopically grafted onto mice. Epifluorescent illumination of the transplanted zone allowed in vivo monitoring of the genetically modified graft. EGFP-positive human skin was present on mice for 22 weeks after grafting. In addition, frozen sections prepared from the grafts displayed consistently strong EGFP-based fluorescence in all epidermal strata at every time point examined. Persistence of transgene expression was further confirmed through EGFP protein immunodetection. Purified EGFP-positive keratinocytes grafted as part of the fibrin-based artificial skin were capable of generating multilayer human epidermis on mice, with well-developed granulosum and corneum strata, and clearly defined rete ridges. Finally, the large proportion of transduced keratinocytes in our grafts allowed us to study, for the first time, the long-term in vivo clonal reconstitution pattern of the regenerated skin. Analysis of the provirus insertion sites indicates that a discrete number of epidermal stem cell clones was responsible for the maintenance of human skin regenerated in NOD/SCID recipients. PMID:12031128

  15. 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. PMID:12606219

  16. The Functional Analysis of Quantum Information Theory

    E-print Network

    Ved Prakash Gupta; Prabha Mandayam; V. S. Sunder

    2015-04-28

    This book is a compilation of notes from a two-week international workshop on the "The Functional Analysis of Quantum Information Theory" that was held at the Institute of Mathematical Sciences during 26/12/2011-06/01/2012. The workshop was devoted to the mathematical framework of quantized functional analysis (QFA), and aimed at illustrating its applications to problems in quantum communication. The lectures were given by Gilles Pisier (Pierre and Marie Curie University and Texas A&M), K.R. Parthasarathy (ISI Delhi), Vern Paulsen (University of Houston), and Andreas Winter (Universitat Autonoma de Barcelona). Topics discussed include Operator Spaces and Completely bounded maps, Schmidt number and Schmidt rank of bipartite entangled states, Operator Systems and Completely Positive Maps, and, Operator Methods in Quantum Information.

  17. The N-Ethyl-N-Nitrosourea-Induced Goldenticket Mouse Mutant Reveals an Essential Function of Sting in the In Vivo Interferon Response to Listeria monocytogenes and Cyclic Dinucleotides ?

    PubMed Central

    Sauer, John-Demian; Sotelo-Troha, Katia; von Moltke, Jakob; Monroe, Kathryn M.; Rae, Chris S.; Brubaker, Sky W.; Hyodo, Mamoru; Hayakawa, Yoshihiro; Woodward, Joshua J.; Portnoy, Daniel A.; Vance, Russell E.

    2011-01-01

    Type I interferons (IFNs) are central regulators of the innate and adaptive immune responses to viral and bacterial infections. Type I IFNs are induced upon cytosolic detection of microbial nucleic acids, including DNA, RNA, and the bacterial second messenger cyclic-di-GMP (c-di-GMP). In addition, a recent study demonstrated that the intracellular bacterial pathogen Listeria monocytogenes stimulates a type I IFN response due to cytosolic detection of bacterially secreted c-di-AMP. The transmembrane signaling adaptor Sting (Tmem173, Mita, Mpys, Eris) has recently been implicated in the induction of type I IFNs in response to cytosolic DNA and/or RNA. However, the role of Sting in response to purified cyclic dinucleotides or during in vivo L. monocytogenes infection has not been addressed. In order to identify genes important in the innate immune response, we have been conducting a forward genetic mutagenesis screen in C57BL/6 mice using the mutagen N-ethyl-N-nitrosourea (ENU). Here we describe a novel mutant mouse strain, Goldenticket (Gt), that fails to produce type I IFNs upon L. monocytogenes infection. By genetic mapping and complementation experiments, we found that Gt mice harbor a single nucleotide variant (T596A) of Sting that functions as a null allele and fails to produce detectable protein. Analysis of macrophages isolated from Gt mice revealed that Sting is absolutely required for the type I interferon response to both c-di-GMP and c-di-AMP. Additionally, Sting is required for the response to c-di-GMP and L. monocytogenes in vivo. Our results provide new functions for Sting in the innate interferon response to pathogens. PMID:21098106

  18. Fracture Analysis of Functionally Graded Materials

    SciTech Connect

    Zhang, Ch.; Gao, X. W.; Sladek, J.; Sladek, V.

    2010-05-21

    This paper reports our recent research works on crack analysis in continuously non-homogeneous and linear elastic functionally graded materials. A meshless boundary element method is developed for this purpose. Numerical examples are presented and discussed to demonstrate the efficiency and the accuracy of the present numerical method, and to show the effects of the material gradation on the crack-opening-displacements and the stress intensity factors.

  19. Optical properties of neonatal skin measured in vivo as a function of age and skin pigmentation

    NASA Astrophysics Data System (ADS)

    Bosschaart, Nienke; Mentink, Rosaline; Kok, Joke H.; van Leeuwen, Ton G.; Aalders, Maurice C. G.

    2011-09-01

    Knowledge of the optical properties of neonatal skin is invaluable when developing new, or improving existing optical techniques for use at the neonatal intensive care. In this article, we present in vivo measurements of the absorption ?a and reduced scattering coefficient ?s' of neonatal skin between 450 and 600 nm and assess the influence of age and skin pigmentation on the optical properties. The optical properties were measured using a spatially resolved, steady state diffuse reflectance spectroscopy setup, combined with a modified spatially resolved diffusion model. The method was validated on phantoms with known values for the absorption and reduced scattering coefficient. Values of ?a and ?s' were obtained from the skin at four different body locations (forehead, sternum, hand, and foot) of 60 neonates with varying gestational age, postnatal age, and skin pigmentation. We found that ?a ranged from 0.02 to 1.25 mm-1 and ?s' was in the range of 1 to 2.8 mm-1 (5th to 95th percentile of the patient population), independent of body location. In contrast to previous studies, no to very weak correlation was observed between the optical properties and gestational maturity, but a strong dependency of the absorption coefficient on postnatal age was found for dark skinned patients.

  20. An in vivo molecular response analysis of colorectal cancer treated with Astragalus membranaceus extract.

    PubMed

    Tseng, Ailun; Yang, Chih-Hsueh; Chen, Chih-Hao; Chen, Chang-Han; Hsu, Shih-Lan; Lee, Mei-Hsien; Lee, Hoong-Chien; Su, Li-Jen

    2016-02-01

    The fact that many chemotherapeutic drugs cause chemoresistance and side effects during the course of colorectal cancer treatment necessitates development of novel cytotoxic agents aiming to attenuate new molecular targets. Here, we show that Astragalus membranaceus (Fischer) Bge. var. mongolicus (Bge.) Hsiao (AM), a traditional Chinese medicine, can inhibit tumor growth in vivo and elucidate the underlying molecular mechanisms. The antitumor effect of AM was assessed on the subcutaneous tumors of human colorectal cancer cell line HCT116 grafted into nude mice. The mice were treated with either water or 500 mg/kg AM once per day, before being sacrificed for extraction of tumors, which were then subjected to microarray expression profiling. The gene expression of the extraction was then profiled using microarray analysis. The identified genes differentially expressed between treated mice and controls reveal that administration of AM suppresses chromosome organization, histone modification, and regulation of macromolecule metabolic process. A separate analysis focused on differentially expressed microRNAs revealing involvement of macromolecule metabolism, and intracellular transport, as well as several cancer signaling pathways. For validation, the input of the identified genes to The Library of Integrated Network-based Cellular Signatures led to many chemopreventive agents of natural origin that produce similar gene expression profiles to that of AM. The demonstrated effectiveness of AM suggests a potential therapeutic drug for colorectal cancer. PMID:26719057

  1. Chromosome mechanics in vivo: quantitative analysis of nonrigid 3D chromosome motion in Drosophila embryos

    NASA Astrophysics Data System (ADS)

    Marshall, Wallace F.; Agard, David A.; Sedat, John W.

    1996-05-01

    Chromosomes are often arranged into specific configurations. One example is the metaphase plate of the Drosophila embryo in which chromosomes are arranged into a parallel bundle. How is this configuration established and maintained? Quantitative analysis of chromosomes motion in vivo should help answer this question by providing a measure of the relevant mechanical properties of the chromosomes themselves. In addition, motion analysis will allow us to study interactions of chromosomes with the mitotic spindle. In order to analyze moving mitotic chromosomes, we acquire time-lapse 3D images of chromosomes in living Drosophila embryos, and then interactively model the chromosome configuration at each time point. A model-based motion estimation algorithm is then applied. From the motion estimate, we can visualize trajectories of different regions on the chromosomes, such as centromeres and telomeres, during metaphase and during prometaphase congression. In addition, quantitative estimates of mechanical properties such as mobility and flexibility can be computed. In this preliminary report we describe computational tools for tracking and visualizing 3D chromosome motion, and for detecting oscillations in position along the mitotic spindle.

  2. Measurement and analysis of chemically changed mineral fibers after experiments in vitro and in vivo.

    PubMed Central

    Spurny, K R

    1983-01-01

    Asbestos, as well as other natural and man-made mineral fibers used for in vitro and in vivo experiments, must be described and defined physically and chemically as exactly as possible before any application. The interactions of fibers with the physical, chemical (air, water, etc.) and biological (cells, tissues, etc.) environments cause important changes in fiber chemistry and crystalline structure. Also, these should be detected as precisely as possible after each experiment. Our recent investigations dealt with the development of a complex analytical system for such measurements and with some applications of these analytical procedures for fibrous material sampled in the environment and from biological materials. Chemical and physical microanalyses of asbestos and glass fibers obtained by environmental sampling (air, water) and from human and animal tissue have shown chemical and crystalline changes in these particles. Scanning electron microscopy, electron microprobe analysis and mass spectrometry analysis were used in these investigations. A partial or total leakage of elements could be observed. The leakage of elements in fibers is of a statistical nature. Some fibers remained chemically unchanged; in some fibers some elements were partially leached; and in some fibers the majority of metallic elements were leached. The potential meaning of this effect is also discussed. Images FIGURE 1. FIGURE 2. FIGURE 4. FIGURE 6. FIGURE 7. FIGURE 7. FIGURE 12. PMID:6315377

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

    PubMed

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

    2015-07-01

    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) received the manualized protocol over 12 weeks. Primary analyses indicated significantly better posttest performance for the treatment group (compared to controls) on 3 of the 4 measures of emotion decoding and encoding and these were maintained at 5-week follow-up. Analyses of secondary measures favored the treatment group for 1 of the 2 measures; specifically, ASD symptoms were significantly lower at posttest and follow-up. PMID:25643864

  4. DNAM-1-based chimeric antigen receptors enhance T cell effector function and exhibit in vivo efficacy against melanoma.

    PubMed

    Wu, Ming-Ru; Zhang, Tong; Alcon, Andre; Sentman, Charles L

    2015-04-01

    Chimeric antigen receptor (CAR) T cell therapies hold great potential for treating cancers, and new CARs that can target multiple tumor types and have the potential to target non-hematological malignancies are needed. In this study, the tumor recognition ability of a natural killer cell-activating receptor, DNAM-1 was harnessed to design CARs that target multiple tumor types. DNAM-1 ligands, PVR and nectin-2, are expressed on primary human leukemia, myeloma, ovarian cancer, melanoma, neuroblastoma, and Ewing sarcoma. DNAM-1 CARs exhibit high tumor cell cytotoxicity but low IFN-? secretion in vitro. In contrast to other CAR designs, co-stimulatory domains did not improve the expression and function of DNAM-1 CARs. A DNAM-1/CD3zeta CAR reduced tumor burden in a murine melanoma model in vivo. In conclusion, DNAM-1-based CARs may have the potential to treat PVR and nectin-2 expressing hematological and solid tumors. PMID:25549845

  5. Synthesis, in vitro, and in vivo evaluation of novel functionalized quaternary ammonium curcuminoids as potential anti-cancer agents.

    PubMed

    Solano, Lucas N; Nelson, Grady L; Ronayne, Conor T; Lueth, Erica A; Foxley, Melissa A; Jonnalagadda, Sravan K; Gurrapu, Shirisha; Mereddy, Venkatram R

    2015-12-15

    Novel functionalized quaternary ammonium curcuminoids have been synthesized from piperazinyl curcuminoids and Baylis-Hillman reaction derived allyl bromides. These molecules are found to be highly water soluble with increased cytotoxicity compared to native curcumin against three cancer cell lines MIAPaCa-2, MDA-MB-231, and 4T1. Preliminary in vivo toxicity evaluation of a representative curcuminoid 5a in healthy mice indicates that this molecule is well tolerated based on normal body weight gains compared to control group. Furthermore, the efficacy of 5a has been tested in a pancreatic cancer xenograft model of MIAPaCa-2 and has been found to exhibit good tumor growth inhibition as a single agent and also in combination with clinical pancreatic cancer drug gemcitabine. PMID:26561365

  6. Rapid experience-dependent plasticity of synapse function and structure in ferret visual cortex in vivo

    E-print Network

    Yu, Hongbo

    The rules by which visual experience influences neuronal responses and structure in the developing brain are not well understood. To elucidate the relationship between rapid functional changes and dendritic spine remodeling ...

  7. Polymer Fiber Probes Enable Optical Control of Spinal Cord and Muscle Function In Vivo

    E-print Network

    Lu, Chi

    Restoration of motor and sensory functions in paralyzed patients requires the development of tools for simultaneous recording and stimulation of neural activity in the spinal cord. In addition to its complex neurophysiology, ...

  8. In Vivo Inflammatory Effects of Ceria Nanoparticles on CD-1 Mouse: Evaluation by Hematological, Histological, and TEM Analysis

    PubMed Central

    Poma, Anna; Ragnelli, Anna Maria; de Lapuente, Joaquin; Ramos, David; Borras, Miquel; Di Gioacchino, Mario; Santucci, Sandro; De Marzi, Laura

    2014-01-01

    The attention on CeO2-NPs environmental and in vivo effects is due to their presence in diesel exhaust and in diesel filters that release a more water-soluble form of ceria NPs, as well as to their use for medical applications. In this work, acute and subacute in vivo toxicity assays demonstrate no lethal effect of these NPs. Anyhow, performing in vivo evaluations on CD-1 mouse systems, we demonstrate that it is even not correct to assert that ceria NPs are harmless for living systems as they can induce status of inflammation, revealed by hematological-chemical-clinical assays as well as histological and TEM microscope observations. TEM analysis showed the presence of NPs in alveolar macrophages. Histological evaluation demonstrated the NPs presence in lungs tissues and this can be explained by assuming their ability to go into the blood stream and lately into the organs (generating inflammation). PMID:25032226

  9. Quantification of Human Brain Metabolites from in Vivo1H NMR Magnitude Spectra Using Automated Artificial Neural Network Analysis

    NASA Astrophysics Data System (ADS)

    Hiltunen, Yrjö; Kaartinen, Jouni; Pulkkinen, Juhani; Häkkinen, Anna-Maija; Lundbom, Nina; Kauppinen, Risto A.

    2002-01-01

    Long echo time (TE=270 ms) in vivo proton NMR spectra resembling human brain metabolite patterns were simulated for lineshape fitting (LF) and quantitative artificial neural network (ANN) analyses. A set of experimental in vivo1H NMR spectra were first analyzed by the LF method to match the signal-to-noise ratios and linewidths of simulated spectra to those in the experimental data. The performance of constructed ANNs was compared for the peak area determinations of choline-containing compounds (Cho), total creatine (Cr), and N-acetyl aspartate (NAA) signals using both manually phase-corrected and magnitude spectra as inputs. The peak area data from ANN and LF analyses for simulated spectra yielded high correlation coefficients demonstrating that the peak areas quantified with ANN gave similar results as LF analysis. Thus, a fully automated ANN method based on magnitude spectra has demonstrated potential for quantification of in vivo metabolites from long echo time spectroscopic imaging.

  10. Over-expression of Arabidopsis thaliana carotenoid hydroxylases individually and in combination with a beta-carotene ketolase provides insight into in vivo functions.

    PubMed

    Kim, Ji-Eun; Cheng, Kimberly M; Craft, Neal E; Hamberger, Björn; Douglas, Carl J

    2010-02-01

    Carotenoids represent a group of widely distributed pigments derived from the general isoprenoid biosynthetic pathway that possess diverse functions in plant primary and secondary metabolism. Modification of alpha- and beta-carotene backbones depends in part on ring hydroxylation. Two ferredoxin-dependent non-heme di-iron monooxygenases (AtB1 and AtB2) that mainly catalyze in vivo beta-carotene hydroxylations of beta,beta-carotenoids, and two heme-containing cytochrome P450 (CYP) monooxygenases (CYP97A3 and CYP97C1) that preferentially hydroxylate the epsilon-ring of alpha-carotene or the beta-ring of beta,epsilon-carotenoids, have been characterized in Arabidopsis by analysis of loss-of-function mutant phenotypes. We further investigated functional roles of both hydroxylase classes in modification of the beta- and epsilon-rings of alpha-carotene and beta-carotene through over-expression of AtB1, CYP97A3, CYP97C1, and the hydroxylase candidate CYP97B3. Since carotenoid hydroxylation is required for generation of ketocarotenoids by the bkt1(CrtO) beta-carotene ketolase, all hydroxylase constructs were also introduced into an Arabidopsis line expressing the Haematococcus pluvalis bkt1 beta-carotene ketolase. Analysis of foliar carotenoid profiles in lines overexpressing the individual hydroxylases indicate a role for CYP97B3 in carotenoid biosynthesis, confirm and extend previous findings of hydroxylase activities based on knock-out mutants, and suggest functions of the multifunctional enzymes in carotenoid biosynthesis. Hydroxylase over-expression in combination with bkt1 did not result in ketocarotenoid accumulation, but instead unexpected patterns of alpha-carotene derivatives, accompanied by a reduction of alpha-carotene, were observed. These data suggest possible interactions between the beta-carotene ketolase bkt1 and the hydroxylases that impact partitioning of carbon flux into different carotenoid branch pathways. PMID:19939422

  11. An adult tissue-specific stem cell in its niche: a gene profiling analysis of in vivo quiescent and activated muscle satellite cells.

    PubMed

    Pallafacchina, Giorgia; François, Stéphanie; Regnault, Béatrice; Czarny, Bertrand; Dive, Vincent; Cumano, Ana; Montarras, Didier; Buckingham, Margaret

    2010-03-01

    The satellite cell of skeletal muscle provides a paradigm for quiescent and activated tissue stem cell states. We have carried out transcriptome analyses on satellite cells purified by flow cytometry from Pax3(GFP/+) mice. We compared samples from adult skeletal muscles where satellite cells are mainly quiescent, with samples from growing muscles or regenerating (mdx) muscles, where they are activated. Analysis of regulation that is shared by both activated states avoids other effects due to immature or pathological conditions. This in vivo profile differs from that of previously analyzed satellite cells activated after cell culture. It reveals how the satellite cell protects itself from damage and maintains quiescence, while being primed for activation on receipt of the appropriate signal. This is illustrated by manipulation of the corepressor Dach1, and by the demonstration that quiescent satellite cells are better protected from oxidative stress than those from mdx or 1-week-old muscles. The quiescent versus in vivo activated comparison also gives new insights into how the satellite cell controls its niche on the muscle fiber through cell adhesion and matrix remodeling. The latter also potentiates growth factor activity through proteoglycan modification. Dismantling the extracellular matrix is important for satellite cell activation when the expression of proteinases is up-regulated, whereas transcripts for their inhibitors are high in quiescent cells. In keeping with this, we demonstrate that metalloproteinase function is required for efficient regeneration in vivo. PMID:19962952

  12. Endogenous cannabinoid system regulates intestinal barrier function in vivo through cannabinoid type 1 receptor activation.

    PubMed

    Zoppi, Silvia; Madrigal, José L M; Pérez-Nievas, Beatriz G; Marín-Jiménez, Ignacio; Caso, Javier R; Alou, Luis; García-Bueno, Borja; Colón, Arturo; Manzanares, Jorge; Gómez-Lus, M Luisa; Menchén, Luis; Leza, Juan C

    2012-03-01

    The deleterious effects of stress on the gastrointestinal tract seem to be mainly mediated by the induction of intestinal barrier dysfunction and subsequent subtle mucosal inflammation. Cannabinoid 1 receptor (CB1R) is expressed in the mammalian gut under physiological circumstances. The aim of this investigation is to study the possible role of CB1R in the maintenance of mucosal homeostasis after stress exposure. CB1R knockout mice (CB1R(-/-)) and their wild-type (WT) counterparts were exposed to immobilization and acoustic (IA) stress for 2 h per day during 4 consecutive days. Colonic protein expression of the inducible forms of the nitric oxide synthase and cyclooxygenase (NOS2 and COX2), IgA production, permeability to (51)Cr-EDTA, and bacterial translocation to mesenteric lymph nodes were evaluated. Stress exposure induced greater expression of proinflammatory enzymes NOS2 and COX2 in colonic mucosa of CB1R(-/-) mice when compared with WT animals. These changes were related with a greater degree of colonic barrier dysfunction in CB1R(-/-) animals determined by 1) a significantly lower IgA secretion, 2) higher paracellular permeability to (51)Cr-EDTA, and 3) higher bacterial translocation, both under basal conditions and after IA stress exposure. Pharmacological antagonism with rimonabant reproduced stress-induced increase of proinflammatory enzymes in the colon described in CB1R(-/-) mice. In conclusion, CB1R exerts a protective role in the colon in vivo through the regulation of intestinal secretion of IgA and paracellular permeability. Pharmacological modulation of cannabinoid system within the gastrointestinal tract might be therapeutically useful in conditions on which intestinal inflammation and barrier dysfunction takes place after exposure to stress. PMID:22135307

  13. Mathematical Analysis of the Pharmacokinetic-Pharmacodynamic (PKPD) Behaviour of Monoclonal Antibodies: Predicting in vivo

    E-print Network

    Derks, Gianne

    Antibodies: Predicting in vivo Potency Philip J. Astona, , Gianne Derksa , Adewale Rajib,a,c , Balaji M the target affinity of a monoclonal antibody and its in vivo potency. The dynamics of the system is described there is no such effect when increasing kon . Thus, for certain monoclonal antibodies, an increase in potency may

  14. Functional analysis of the DNA-stimulated ATPase domain of yeast SWI2/SNF2.

    PubMed Central

    Richmond, E; Peterson, C L

    1996-01-01

    The yeast SWI2/SNF2 polypeptide is a subunit of the SWI/SNF protein complex that is required for many transcriptional activators to function in a chromatin context. SWI2 is believed to be the founding member of a new subfamily of DNA-stimulated ATPases/DNA helicases that includes proteins that function in DNA repair (RAD5, RAD16, ERCC6), recombination (RAD54), transcription (MOT1, ISWI, brm, BRG1, hBRM) and cell cycle control (STH1). We have created a set of 16 mutations within the SWI2 ATPase domain and have analyzed the functional consequences of these mutations in vivo. We have identified residues within each of the seven ATPase motifs that are required for SWI2 function. We have also identified crucial residues that are interspersed between the known ATPase motifs. In contrast, we identify other highly conserved residues that appear to be dispensable for SWI2 function. We also find that single amino acid changes in ATPase motifs IV and VI lead to a dominant negative phenotype. None of the 12 SWI2 mutations that disrupt SWI2 activity in vivo alter the assembly of the SWI/SNF complex. These studies provide an invaluable framework for biochemical analysis of the SWI2 ATPase and for functional analysis of other SWI2 family members. PMID:8871545

  15. Depletion of retinal dopamine does not affect the ERG b-wave increment threshold function in goldfish in vivo.

    PubMed

    Lin, Z S; Yazulla, S

    1994-01-01

    Increment threshold functions of the electroretinogram (ERG) b-wave were obtained from goldfish using an in vivo preparation to study intraretinal mechanisms underlying the increase in perceived brightness induced by depletion of retinal dopamine by 6-hydroxydopamine (6-OHDA). Goldfish received unilateral intraocular injections of 6-OHDA plus pargyline on successive days. Depletion of retinal dopamine was confirmed by the absence of tyrosine-hydroxylase immunoreactivity at 2 to 3 weeks postinjection as compared to sham-injected eyes from the same fish. There was no difference among normal, sham-injected or 6-OHDA-injected eyes with regard to ERG waveform, intensity-response functions or increment threshold functions. Dopamine-depleted eyes showed a Purkinje shift, that is, a transition from rod-to-cone dominated vision with increasing levels of adaptation. We conclude (1) dopamine-depleted eyes are capable of photopic vision; and (2) the ERG b-wave is not diagnostic for luminosity coding at photopic backgrounds. We also predict that (1) dopamine is not required for the transition from scotopic to photopic vision in goldfish; (2) the ERG b-wave in goldfish is influenced by chromatic interactions; (3) horizontal cell spinules, though correlated with photopic mechanisms in the fish retina, are not necessary for the transition from scotopic to photopic vision; and (4) the OFF pathway, not the ON pathway, is involved in the action of dopamine on luminosity coding in the retina. PMID:7918220

  16. Odorant Metabolism Analysis by an Automated Ex Vivo Headspace Gas-Chromatography Method.

    PubMed

    Faure, Philippe; Legendre, Aričle; Hanser, Hassan-Ismail; Andriot, Isabelle; Artur, Yves; Guichard, Elisabeth; Coureaud, Gérard; Heydel, Jean-Marie

    2016-01-01

    In the olfactory epithelium (OE), odorant metabolizing enzymes have the dual function of volatile component detoxification and active clearance of odorants from the perireceptor environment to respectively maintain the integrity of the tissues and the sensitivity of the detection. Although emphasized by recent studies, this enzymatic mechanism is poorly documented in mammals. Thus, olfactory metabolism has been characterized mainly in vitro and for a limited number of odorants. The automated ex vivo headspace gas-chromatography method that was developed here was validated to account for odorant olfactory metabolism. This method easily permits the measurement of the fate of an odorant in the OE environment, taking into account the odorant gaseous state and the cellular structure of the tissue, under experimental conditions close to physiological conditions and with a high reproducibility. We confirmed here our previous results showing that a high olfactory metabolizing activity of the mammary pheromone may be necessary to maintain a high level of sensitivity toward this molecule, which is critical for newborn rabbit survival. More generally, the method that is presented here may permit the screening of odorants metabolism alone or in mixture or studying the impact of aging, pathology, polymorphism or inhibitors on odorant metabolism. PMID:26446453

  17. 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.

  18. Heparin inhibition of von Willebrand factor-dependent platelet function in vitro and in vivo.

    PubMed Central

    Sobel, M; McNeill, P M; Carlson, P L; Kermode, J C; Adelman, B; Conroy, R; Marques, D

    1991-01-01

    The intravenous administration of heparin to patients before open heart surgery reduced ristocetin cofactor activity by 58% (P less than 0.01, t test), and this impairment of von Willebrand factor-dependent platelet function was closely related to plasma heparin levels (r2 = 0.9), but not to plasma von Willebrand factor (vWF) levels. We hypothesized that heparin may inhibit vWF-dependent platelet hemostatic functions by directly binding vWF in solution and interfering with vWF-GpIb binding. Using the in vitro techniques of ristocetin-induced platelet agglutination, fluorescent flow cytometric measurement of vWF-platelet binding, and conventional radioligand binding assays we observed that heparin inhibited both vWF-dependent platelet function and vWF-platelet binding in a parallel and dose-dependent manner. Heparin also inhibited platelet agglutination induced by bovine vWF and inhibited the binding of human asialo-vWF to platelets in ristocetin-free systems. The inhibitory potency of heparin was not dependent upon its affinity for antithrombin III, but was molecular weight dependent: homogeneous preparations of lower molecular weight were less inhibitory. Heparin impairment of vWF function may explain why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin. PMID:2022745

  19. IN VITRO/IN VIVO COMPARISON OF YOLK SAC FUNCTION AND EMBRYO DEVELOPMENT

    EPA Science Inventory

    Yolk sac function and development of rat embryos grown in vitro for 24 hrs starting on day 10.5 were compared to those of embryos grown in utero. he embryos grown in vitro had significantly fewer somites, shorter crown-rump length and smaller yolk sac diameter when compared to th...

  20. 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 ?M 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 ?mol/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 ?mol/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

  1. Spectral domain optical coherence tomography for ex vivo brain tumor analysis

    NASA Astrophysics Data System (ADS)

    Lenz, Marcel; Krug, Robin; Jaedicke, Volker; Stroop, Ralf; Schmieder, Kirsten; Hofmann, Martin R.

    2015-07-01

    Non-contact imaging methods to distinguish between healthy tissue and brain tumor tissue during surgery would be highly desirable but are not yet available. Optical Coherence Tomography (OCT) is a non-invasive imaging technology with a resolution around 1-15 ?m and a penetration depth of 1-2 mm that may satisfy the demands. To analyze its potential, we measured ex vivo human brain tumor tissue samples from 10 patients with a Spectral Domain OCT system (Thorlabs Callisto: center wavelength of 930 nm) and compared the results with standard histology. In detail, three different measurements were made for each sample. First the sample was measured directly after surgery. Then it was embedded in paraffin (also H and E staining) and examined for the second time. At last, the slices of each paraffin block cut by the pathology were measured. Each time a B-scan was created and for a better comparison with the histology a 3D image was generated, in order to get the corresponding en face images. In both, histopathological diagnosis and the analysis of the OCT images, different types of brain tumor showed difference in structure. This has been affirmed by two blinded investigators. Nevertheless the difference between two images of samples taken directly after surgery is less distinct. To enhance the contrast in the images further, we employ Spectroscopic OCT and pattern recognition algorithms and compare these results to the histopathological standard.

  2. Flux analysis of cholesterol biosynthesis in vivo reveals multiple tissue and cell-type specific pathways

    PubMed Central

    Mitsche, Matthew A; McDonald, Jeffrey G; Hobbs, Helen H; Cohen, Jonathan C

    2015-01-01

    Two parallel pathways produce cholesterol: the Bloch and Kandutsch-Russell pathways. Here we used stable isotope labeling and isotopomer analysis to trace sterol flux through the two pathways in mice. Surprisingly, no tissue used the canonical K–R pathway. Rather, a hybrid pathway was identified that we call the modified K–R (MK–R) pathway. Proportional flux through the Bloch pathway varied from 8% in preputial gland to 97% in testes, and the tissue-specificity observed in vivo was retained in cultured cells. The distribution of sterol isotopomers in plasma mirrored that of liver. Sterol depletion in cultured cells increased flux through the Bloch pathway, whereas overexpression of 24-dehydrocholesterol reductase (DHCR24) enhanced usage of the MK–R pathway. Thus, relative use of the Bloch and MK–R pathways is highly variable, tissue-specific, flux dependent, and epigenetically fixed. Maintenance of two interdigitated pathways permits production of diverse bioactive sterols that can be regulated independently of cholesterol. DOI: http://dx.doi.org/10.7554/eLife.07999.001 PMID:26114596

  3. A functional analysis of hair pulling.

    PubMed Central

    Rapp, J T; Miltenberger, R G; Galensky, T L; Ellingson, S A; Long, E S

    1999-01-01

    We experimentally assessed the functions of hair pulling and hair manipulation of a 19-year-old woman (Kris) with moderate mental retardation and cerebral palsy. In Phase 1 a functional analysis revealed that Kris pulled and manipulated hair for the greatest amount of time in the alone condition, suggesting that the behaviors were maintained by some form of automatic reinforcement (Vaughan & Michael, 1982). In Phase 2 we assessed the nature of the sensory stimulation that maintained hair pulling by providing continuous access to previously pulled or cut hair and, thereafter, by having Kris wear a rubber glove. The results suggested that hair pulling was maintained by digital-tactile stimulation (automatic positive reinforcement). These findings are discussed, and recommendations for further analyses of automatically reinforced habit behaviors are provided. PMID:10513028

  4. Effects upon in vivo nicotine metabolism reveal functional variation in FMO3 associated with cigarette consumption

    PubMed Central

    Bloom, A. Joseph; Murphy, Sharon E.; Martinez, Maribel; von Weymarn, Linda B.; Bierut, Laura J.; Goate, Alison

    2015-01-01

    Background Flavin-containing monooxygenases (FMO) catalyze the metabolism of nucleophilic heteroatom containing drugs and xenobiotics including nicotine. Rare mutations in FMO3 are responsible for defective N-oxygenation of dietary trimethylamine leading to trimethylaminuria, and common genetic variation in FMO3 has been linked to interindividual variability in metabolic function that may be substrate specific. Methods A genetic model of CYP2A6 function is used as a covariate to reveal functional polymorphism in FMO3 that indirectly influences the ratio of deuterated nicotine metabolized to cotinine following oral administration. The association is tested between FMO3 haplotype and cigarette consumption in a set of nicotine dependent smokers. Results FMO3 haplotype, based on all common coding variants in Europeans, significantly predicts nicotine metabolism and accounts for approximately 2% of variance in the apparent percent of nicotine metabolized to cotinine. The metabolic ratio is not associated with FMO2 haplotype or an FMO1 expression quantitative trait locus (eQTL). Cross validation demonstrates calculated FMO3 haplotype parameters to be robust and significantly improve the predictive nicotine metabolism model over CYP2A6 genotype alone. Functional classes of FMO3 haplotypes, as determined by their influence on nicotine metabolism to cotinine, are also significantly associated with cigarettes per day (CPD) in nicotine dependent European Americans (n=1,025, p=0.04), and significantly interact (p=0.016) with CYP2A6 genotype to predict CPD. Conclusion These findings suggest that common polymorphisms in FMO3 influence nicotine clearance, and that these genetic variants in turn influence cigarette consumption. PMID:23211429

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

    PubMed Central

    Preuße, Kristina; Tveriakhina, Lena; Schuster-Gossler, Karin; Gaspar, Cláudia; Rosa, Alexandra Isabel; Henrique, Domingos; Gossler, Achim; Stauber, Michael

    2015-01-01

    Notch signalling is a fundamental pathway that shapes the developing embryo and sustains adult tissues by direct communication between ligand and receptor molecules on adjacent cells. Among the ligands are two Delta paralogues, DLL1 and DLL4, that are conserved in mammals and share a similar structure and sequence. They activate the Notch receptor partly in overlapping expression domains where they fulfil redundant functions in some processes (e.g. maintenance of the crypt cell progenitor pool). In other processes, however, they appear to act differently (e.g. maintenance of foetal arterial identity) raising the questions of how similar DLL1 and DLL4 really are and which mechanism causes the apparent context-dependent divergence. By analysing mice that conditionally overexpress DLL1 or DLL4 from the same genomic locus (Hprt) and mice that express DLL4 instead of DLL1 from the endogenous Dll1 locus (Dll1Dll4ki), we found functional differences that are tissue-specific: while DLL1 and DLL4 act redundantly during the maintenance of retinal progenitors, their function varies in the presomitic mesoderm (PSM) where somites form in a Notch-dependent process. In the anterior PSM, every cell expresses both Notch receptors and ligands, and DLL1 is the only activator of Notch while DLL4 is not endogenously expressed. Transgenic DLL4 cannot replace DLL1 during somitogenesis and in heterozygous Dll1Dll4ki/+ mice, the Dll1Dll4ki allele causes a dominant segmentation phenotype. Testing several aspects of the complex Notch signalling system in vitro, we found that both ligands have a similar trans-activation potential but that only DLL4 is an efficient cis-inhibitor of Notch signalling, causing a reduced net activation of Notch. These differential cis-inhibitory properties are likely to contribute to the functional divergence of DLL1 and DLL4. PMID:26114479

  6. In vivo functional photoacoustic microscopy of cutaneous microvasculature in human skin

    NASA Astrophysics Data System (ADS)

    Favazza, Christopher P.; Cornelius, Lynn A.; Wang, Lihong V.

    2011-02-01

    Microcirculation is an important component of the cardiovascular system and can be used to assess systemic cardiovascular health. Numerous studies have investigated cutaneous microcirculation as an indicator of cardiovascular related diseases. Such research has shown promising results; however, there are many limitations regarding the employed measurement techniques, such as poor depth and spatial resolution and measurement versatility. Here we show the results of functional cutaneous microvascular experiments measured with photoacoustic microscopy, which provides high spatial resolution and multiparameter measurements. In a set of experiments, microvascular networks located in the palms of volunteers were perturbed by periodic ischemic events, and the subsequent hemodynamic response to the stimulus was recorded. Results indicate that during periods of arterial occlusion, the relative oxygen saturation of the capillary vessels decreased below resting levels, and temporarily increased above resting levels immediately following the occlusion. Furthermore, a hyperemic reaction to the occlusions was measured, and the observation agreed well with similar measurements using more conventional imaging techniques. Due to its exceptional capability to functionally image vascular networks with high spatial resolution, photoacoustic microscopy could be a beneficial biomedical tool to assess microvascular functioning and applied to patients with diseases that affect cardiovascular health.

  7. Functional characterization of dopamine transporter in vivo using Drosophila melanogaster behavioral assays

    PubMed Central

    Ueno, Taro; Kume, Kazuhiko

    2014-01-01

    Dopamine mediates diverse functions such as motivation, reward, attention, learning/memory and sleep/arousal. Recent studies using model organisms including the fruit fly, have elucidated various physiological functions of dopamine, and identified specific neural circuits for these functions. Flies with mutations in the Drosophila dopamine transporter (dDAT) gene show enhanced dopamine signaling, and short sleep and memory impairment phenotypes. However, understanding the mechanism by which dopamine signaling causes these phenotypes requires an understanding of the dynamics of dopamine release. Here we report the effects of dDAT expression on behavioral traits. We show that dDAT expression in a subset of dopaminergic neurons is sufficient for normal sleep. dDAT expression in other cell types such as Kenyon cells and glial cells can also rescue the short sleep phenotype of dDAT mutants. dDAT mutants also show a down-regulation of the D1-like dopamine receptor dDA1, and this phenotype is rescued when dDAT is expressed in the same cell types in which it rescues sleep. On the other hand, dDAT overexpression in mushroom bodies, which are the target of memory forming dopamine neurons, abolishes olfactory aversive memory. Our data demonstrate that expression of extrasynaptic dopamine transporters can rescue some aspects of dopamine signaling in dopamine transporter mutants. These results provide novel insights into regulatory systems that modulate dopamine signaling. PMID:25232310

  8. In vitro and in vivo studies of macrophage functions in amebiasis.

    PubMed

    Denis, M; Chadee, K

    1988-12-01

    Experimental intrahepatic inoculation of the gerbil with Entamoeba histolytica trophozoites was used as a model of liver amebiasis to study the cellular immune response elicited by the parasite. It was shown that abscess-derived macrophages (5 to 20 days old) were deficient in their capacity to develop a respiratory burst, to secrete and express membrane-bound interleukin-1-like activity, and to kill E. histolytica trophozoites as well as to respond to lymphokines in vitro. However, macrophages isolated from the spleen and peritoneal cavities from the same infected animals were not significantly down regulated in these functions. Splenocytes from infected gerbils were shown to develop a strong responsiveness to amebic antigen, whereas their response to concanavalin A was suppressed. Crude E. histolytica extracts or conditioned medium down regulated murine BALB/c macrophage accessory and effector cell functions in vitro in a manner similar to abscess-derived macrophages, whereas crude extracts of the nonvirulent E. histolytica-like Laredo strain did not. Our results indicate that intrinsic or secreted products or both from E. histolytica are actively regulating macrophage functions at the abscess site and can possibly mediate other immunoregulatory mechanisms at distant targets. PMID:2903124

  9. Automation and uncertainty analysis of a method for in-vivo range verification in particle therapy

    NASA Astrophysics Data System (ADS)

    Frey, K.; Unholtz, D.; Bauer, J.; Debus, J.; Min, C. H.; Bortfeld, T.; Paganetti, H.; Parodi, K.

    2014-10-01

    We introduce the automation of the range difference calculation deduced from particle-irradiation induced ?+-activity distributions with the so-called most-likely-shift approach, and evaluate its reliability via the monitoring of algorithm- and patient-specific uncertainty factors. The calculation of the range deviation is based on the minimization of the absolute profile differences in the distal part of two activity depth profiles shifted against each other. Depending on the workflow of positron emission tomography (PET)-based range verification, the two profiles under evaluation can correspond to measured and simulated distributions, or only measured data from different treatment sessions. In comparison to previous work, the proposed approach includes an automated identification of the distal region of interest for each pair of PET depth profiles and under consideration of the planned dose distribution, resulting in the optimal shift distance. Moreover, it introduces an estimate of uncertainty associated to the identified shift, which is then used as weighting factor to ‘red flag’ problematic large range differences. Furthermore, additional patient-specific uncertainty factors are calculated using available computed tomography (CT) data to support the range analysis. The performance of the new method for in-vivo treatment verification in the clinical routine is investigated with in-room PET images for proton therapy as well as with offline PET images for proton and carbon ion therapy. The comparison between measured PET activity distributions and predictions obtained by Monte Carlo simulations or measurements from previous treatment fractions is performed. For this purpose, a total of 15 patient datasets were analyzed, which were acquired at Massachusetts General Hospital and Heidelberg Ion-Beam Therapy Center with in-room PET and offline PET/CT scanners, respectively. Calculated range differences between the compared activity distributions are reported in a 2D map in beam-eye-view. In comparison to previously proposed approaches, the new most-likely-shift method shows more robust results for assessing in-vivo the range from strongly varying PET distributions caused by differing patient geometry, ion beam species, beam delivery techniques, PET imaging concepts and counting statistics. The additional visualization of the uncertainties and the dedicated weighting strategy contribute to the understanding of the reliability of observed range differences and the complexity in the prediction of activity distributions. The proposed method promises to offer a feasible technique for clinical routine of PET-based range verification.

  10. Biosignals Analysis for Kidney Function Effect Analysis of Fennel Aromatherapy

    PubMed Central

    Kim, Bong-Hyun; Cho, Dong-Uk; Seo, Ssang-Hee

    2015-01-01

    Human effort in order to enjoy a healthy life is diverse. IT technology to these analyzes, the results of development efforts, it has been applied. Therefore, I use the care and maintenance diagnostic health management and prevention than treatment. In particular, the aromatherapy treatment easy to use without the side effects there is no irritation, are widely used in modern society. In this paper, we measured the aroma effect by applying a biosignal analysis techniques; an experiment was performed to analyze. In particular, we design methods and processes of research based on the theory aroma that affect renal function. Therefore, in this paper, measuring the biosignals and after fennel aromatherapy treatment prior to the enforcement of the mutual comparison, through the analysis, studies were carried out to analyze the effect of fennel aromatherapy therapy on kidney function. PMID:25977696

  11. Biosignals analysis for kidney function effect analysis of fennel aromatherapy.

    PubMed

    Kim, Bong-Hyun; Cho, Dong-Uk; Seo, Ssang-Hee

    2015-01-01

    Human effort in order to enjoy a healthy life is diverse. IT technology to these analyzes, the results of development efforts, it has been applied. Therefore, I use the care and maintenance diagnostic health management and prevention than treatment. In particular, the aromatherapy treatment easy to use without the side effects there is no irritation, are widely used in modern society. In this paper, we measured the aroma effect by applying a biosignal analysis techniques; an experiment was performed to analyze. In particular, we design methods and processes of research based on the theory aroma that affect renal function. Therefore, in this paper, measuring the biosignals and after fennel aromatherapy treatment prior to the enforcement of the mutual comparison, through the analysis, studies were carried out to analyze the effect of fennel aromatherapy therapy on kidney function. PMID:25977696

  12. Markov processes Stochastic analysis for additive functionals Geometry of rough spaces Stochastic analysis for Markov processes

    E-print Network

    Weber, Stefan

    Markov processes Stochastic analysis for additive functionals Geometry of rough spaces Stochastic analysis for Markov processes Michael Hinz Bielefeld University Colloquium Stochastic Analysis, Leibniz for Markov processes #12;Markov processes Stochastic analysis for additive functionals Geometry of rough

  13. In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters

    PubMed Central

    Palmer, Gregory M; Fontanella, Andrew N; Shan, Siqing; Hanna, Gabi; Zhang, Guoqing; Fraser, Cassandra L; Dewhirst, Mark W

    2012-01-01

    Optical techniques for functional imaging in mice have a number of key advantages over other common imaging modalities such as magnetic resonance imaging, positron emission tomography or computed tomography, including high resolution, low cost and an extensive library of available contrast agents and reporter genes. A major challenge to such work is the limited penetration depth imposed by tissue turbidity. We describe a window chamber technique by which these limitations can be avoided. This facilitates the study of a wide range of processes, with potential endpoints including longitudinal gene expression, vascular remodeling and angiogenesis, and tumor growth and invasion. We further describe several quantitative imaging and analysis techniques for characterizing in vivo fluorescence properties and functional endpoints, including vascular morphology and oxygenation. The procedure takes ~2 h to complete, plus up to several weeks for tumor growth and treatment procedures. PMID:21886101

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

    PubMed Central

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

    2015-01-01

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

  15. A novel single walled carbon nanotube (SWCNT) functionalization agent facilitating in vivo combined chemo/thermo therapy.

    PubMed

    Zhang, Liwen; Rong, Pengfei; Chen, Minglong; Gao, Shi; Zhu, Lei

    2015-10-21

    Carbon nanotubes (CNTs) have shown intriguing applications in biotechnological and biomedical fields due to their unique shape and properties. However, the fact that unmodified CNTs are prone to aggregation, stunts CNTs applications under physiological conditions. In this research, we found that as little as 1/5th the single walled carbon nanotube (SWCNT) weight of Evans Blue (EB) is capable of dispersing SWCNT as well as facilitating SWCNT functionalization. In view of the binding between EB and albumin, the yielding product (SWCNT/EB) demonstrated extreme stability for weeks under physiological conditions and it can be endowed with a therapeutic ability by simply mixing SWCNT/EB with an albumin based drug. Specifically, the formed SWCNT/EB/albumin/PTX nanocomplex exhibits strong near-infrared (NIR) absorbance, and can serve as an agent for chemo/thermal therapeutic purposes. Our in vivo result reveals that SWCNT/EB/albumin/PTX after being administered into the MDA-MB-435 tumor would effectively ablate the tumor by chemo and photothermal therapy. Such a combined treatment strategy provides remarkable therapeutic outcomes in restraining tumor growth compared to chemo or photothermal therapy alone. Overall, our strategy of dispersing SWCNTs by EB can be used as a platform for carrying other drugs or functional genes with the aid of albumin to treat diseases. The present study opens new opportunities in surface modification of SWCNTs for future clinical disease treatment. PMID:26234690

  16. Effects of GC7101, a Novel Prokinetic Agent on Gastric Motor Function: Ex Vivo Study

    PubMed Central

    Jung, Da Hyun; Choi, Eun Ju; Jeon, Han Ho; Lee, Young Ho; Park, Hyojin

    2014-01-01

    Background/Aims GC7101, an extract of Lonicera Flos, is a novel developing drug for reflux esophagitis and functional dyspepsia. However, the drug’s exact pharmacological mechanism of action remains unclear. This study assessed the effects of GC7101 on gastrointestinal (GI) motor function. Methods We used male guinea pigs to evaluate the effects of GC7101 on GI motility. The contraction of antral circular muscle in the presence of different doses of GC7101 was measured in a tissue bath. The prokinetic effects of GC7101 were tested using the charcoal transit assay from the pylorus to the most distal point of migration of charcoal mixture. To clarify the mechanism of action of GC7101, atropine, dopamine and the selective 5-hydroxytryptamine 4 receptor antagonist, GR113808 were used. Results The maximal amplitude of circular muscle contraction was induced by 5 mg mL?1 GC7101. The area under the curve of contraction was significantly increased at 5 mg mL?1 GC7101. Addition of 10?6 M atropine, 10?8 M dopamine or 10?7 M GR 113808 to GC7101 5 mg mL?1 decreased the amplitude and area under curve compared to GC7101 5 mg mL?1 alone. GC7101 accelerated GI transit in a dose dependent manner except 100 mg kg?1. Delayed GI transit caused by atropine, dopamine and GR 113808 was restored by GC7101 50 mg kg?1. Conclusions GC7101, an extract of Lonicera Flos, exerts a gastric prokinetic effect in guinea pig through cholinergic, antidopaminergic and serotonergic mechanisms. Therefore, GC7101 might be a novel drug for the treatment of functional dyspepsia. PMID:25273117

  17. In vivo functional mapping of the conserved protein domains within murine Themis1.

    PubMed

    Zvezdova, Ekaterina; Lee, Jan; El-Khoury, Dalal; Barr, Valarie; Akpan, Itoro; Samelson, Lawrence; Love, Paul E

    2014-09-01

    Thymocyte development requires the coordinated input of signals that originate from numerous cell surface molecules. Although the majority of thymocyte signal-initiating receptors are lineage-specific, most trigger 'ubiquitous' downstream signaling pathways. T-lineage-specific receptors are coupled to these signaling pathways by lymphocyte-restricted adapter molecules. We and others recently identified a new putative adapter protein, Themis1, whose expression is largely restricted to the T lineage. Mice lacking Themis1 exhibit a severe block in thymocyte development and a striking paucity of mature T cells revealing a critical role for Themis1 in T-cell maturation. Themis1 orthologs contain three conserved domains: a proline-rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine-containing globular (CABIT) domain. In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1(-/-) progenitor cells. The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function. PMID:24935457

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

    PubMed

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

    2014-04-01

    Lipocalins are small extracellular proteins mostly described as lipid carriers. The Drosophila lipocalin NLaz (neural Lazarillo) modulates the IIS pathway and regulates longevity, stress resistance, and behavior. Here, we test whether a native hydrophobic pocket structure is required for NLaz to perform its functions. We use a point mutation altering the binding pocket (NLaz(L130R)) and control mutations outside NLaz binding pocket. Tryptophan fluorescence titration reveals that NLaz(L130R) loses its ability to bind ergosterol and the pheromone 7(z)-tricosene but retains retinoic acid binding. Using site-directed transgenesis in Drosophila, we test the functionality of the ligand binding-altered lipocalin at the organism level. NLaz-dependent life span reduction, oxidative stress and starvation sensitivity, aging markers accumulation, and deficient courtship are rescued by overexpression of NLaz(WT), but not of NLaz(L130R). Transcriptional responses to aging and oxidative stress show a large set of age-responsive genes dependent on the integrity of NLaz binding pocket. Inhibition of IIS activity and modulation of oxidative stress and infection-responsive genes are binding pocket-dependent processes. Control of energy metabolites on starvation appears to be, however, insensitive to the modification of the NLaz binding pocket. PMID:24361577

  19. In vivo effects of Aspalathus linearis (rooibos) on male rat reproductive functions.

    PubMed

    Opuwari, C S; Monsees, T K

    2014-10-01

    Aspalathus linearis (rooibos tea) may improve sperm function owing to its antioxidant properties. To test this hypothesis, male rats were given 2% or 5% rooibos tea for 52 days. No significant alterations were observed in body and reproductive organs weight, serum antioxidant capacity and testosterone level. Seminiferous tubules displayed complete spermatogenesis. However, a significant (P < 0.05) decrease in tubule diameter and germinal epithelial height was observed. Epithelial height of caput epididymides showed a significant increase. Unfermented rooibos significantly enhanced sperm concentration, viability and motility. Fermented rooibos also significantly improved sperm vitality (P < 0.01), but caused a significant increase in spontaneous acrosome reaction (P < 0.05), whereas unfermented did not. Creatinine was significantly enhanced in all treated rats, consistent with significant higher kidney weights. Rooibos significantly reduced alanine transaminase level, while 2% fermented rooibos significantly decreased aspartate transaminase level (P < 0.01). In conclusion, treatment with rooibos improved sperm concentration, viability and motility, which might be attributed to its high level of antioxidants. However, prolonged exposure of rooibos might result in subtle structural changes in the male reproductive system and may induce acrosome reaction, which can impair fertility. Intake of large amounts of rooibos may also harm liver and kidney function. PMID:24007336

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

    NASA Astrophysics Data System (ADS)

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

    Ultra-short pulsed laser (USPL) systems for dental application have overcome many of their initial disadvantages. However, a problem that has not yet been addressed and solved is the beam delivery into the oral cavity. The functional system that is introduced in this study includes an articulated mirror arm, a scanning system as well as a handpiece, allowing for freehand preparations with ultra-short laser pulses. As laser source an Nd:YVO4 laser is employed, emitting pulses with a duration of tp < 10 ps at a repetition rate of up to 500 kHz. The centre wavelength is at 1064 nm and the average output power can be tuned up to 9 W. The delivery system consists of an articulated mirror arm, to which a scanning system and a custom made handpiece are connected, including a 75 mm focussing lens. The whole functional system is compact in size and moveable. General characteristics like optical losses and ablation rate are determined and compared to results employing a fixed setup on an optical table. Furthermore classical treatment procedures like cavity preparation are being demonstrated on mammoth ivory. This study indicates that freehand preparation employing an USPL system is possible but challenging, and accompanied by a variety of side-effects. The ablation rate with fixed handpiece is about 10 mm3/min. Factors like defocussing and blinding affect treatment efficiency. Laser sources with higher average output powers might be needed in order to reach sufficient preparation speeds.

  1. Analysis of In-Vivo LacR-Mediated Gene Repression Based on the Mechanics of DNA Looping

    PubMed Central

    Zhang, Yongli; McEwen, Abbye E.; Crothers, Donald M.; Levene, Stephen D.

    2006-01-01

    Interactions of E. coli lac repressor (LacR) with a pair of operator sites on the same DNA molecule can lead to the formation of looped nucleoprotein complexes both in vitro and in vivo. As a major paradigm for loop-mediated gene regulation, parameters such as operator affinity and spacing, repressor concentration, and DNA bending induced by specific or non-specific DNA-binding proteins (e.g., HU), have been examined extensively. However, a complete and rigorous model that integrates all of these aspects in a systematic and quantitative treatment of experimental data has not been available. Applying our recent statistical-mechanical theory for DNA looping, we calculated repression as a function of operator spacing (58–156 bp) from first principles and obtained excellent agreement with independent sets of in-vivo data. The results suggest that a linear extended, as opposed to a closed v-shaped, LacR conformation is the dominant form of the tetramer in vivo. Moreover, loop-mediated repression in wild-type E. coli strains is facilitated by decreased DNA rigidity and high levels of flexibility in the LacR tetramer. In contrast, repression data for strains lacking HU gave a near-normal value of the DNA persistence length. These findings underscore the importance of both protein conformation and elasticity in the formation of small DNA loops widely observed in vivo, and demonstrate the utility of quantitatively analyzing gene regulation based on the mechanics of nucleoprotein complexes. PMID:17205140

  2. Fourier Analysis and Autocorrelation Function Applied to Periodical Nanostructures

    E-print Network

    Rockett, Angus

    Fourier Analysis and Autocorrelation Function Applied to Periodical Nanostructures E. Cruz Microscopy (AFM) Image Fast Fourier Transformation Autocorrelation Function(AC) Angular Distribution] Fourier Analysis: analytical and geometrical aspects, Bray William O ed. New York: Marcel Dekker, 1994

  3. EFFECT OF OIL COMBUSTION PARTICLE BIOAVAILABLE CONSTITUENTS ON EX VIVO VASCULAR FUNCTION OF AORTAS RECOVERED FROM NORMAL AND TYPE 2 DIABETIC RATS

    EPA Science Inventory

    Effect of Oil Combustion Particle Bioavailable Constituents on Ex Vivo Vascular Function of Aortae Recovered from Healthy and Early Type 2 Diabetic Rats
    KL Dreher1, SE Kelly2, SD Proctor2, and JC Russell2. 1National Health and Environmental Effects Laboratory, US EPA, RTP, NC;...

  4. Supplementary Figure 1. In vivo functional properties of neurons in the EM imaged volume. (Top) Cell-based orientation preference map in mouse visual cortex.

    E-print Network

    Reid, R. Clay

    . Correspondence between in vivo fluorescence anatomy and electron microscopy throughout the EM volume. Merged, with reconstructions of each functionally characterized cell. Horizontal grey bars bound the SUPPLEMENTARY anatomy as in Fig. 1c (red: blood vessels or astrocytes, green: OGB or YFP). Blood vessels and astrocytes

  5. Analysis of Single Locus Trajectories for Extracting In Vivo Chromatin Tethering Interactions

    E-print Network

    Amitai, Assaf

    Is it possible to extract tethering forces applied on chromatin from the statistics of a single locus trajectories imaged in vivo? Chromatin fragments interact with many partners such as the nuclear membrane, other chromosomes ...

  6. In vivo boron-10 analysis for the pre-screening of compounds for BNCS

    E-print Network

    Zhu, Xuping, 1970-

    2004-01-01

    An in vivo boron-10 screening technique was developed to analyze the boron biodistribution in a rabbit knee for the pre-screening of compounds for Boron Neutron Capture Synovectomy (BNCS). Three approaches were investigated: ...

  7. Comprehensive multilevel in vivo and in vitro analysis of heart rate fluctuations in mice by ECG telemetry and electrophysiology.

    PubMed

    Fenske, Stefanie; Pröbstle, Rasmus; Auer, Franziska; Hassan, Sami; Marks, Vanessa; Pauza, Danius H; Biel, Martin; Wahl-Schott, Christian

    2016-01-01

    The normal heartbeat slightly fluctuates around a mean value; this phenomenon is called physiological heart rate variability (HRV). It is well known that altered HRV is a risk factor for sudden cardiac death. The availability of genetic mouse models makes it possible to experimentally dissect the mechanism of pathological changes in HRV and its relation to sudden cardiac death. Here we provide a protocol that allows for a comprehensive multilevel analysis of heart rate (HR) fluctuations. The protocol comprises a set of techniques that include in vivo telemetry and in vitro electrophysiology of intact sinoatrial network preparations or isolated single sinoatrial node (SAN) cells. In vitro preparations can be completed within a few hours, with data acquisition within 1 d. In vivo telemetric ECG requires 1 h for surgery and several weeks for data acquisition and analysis. This protocol is of interest to researchers investigating cardiovascular physiology and the pathophysiology of sudden cardiac death. PMID:26658468

  8. Functional Optical Coherence Tomography Enables In Vivo Physiological Assessment of Retinal Rod and Cone Photoreceptors

    NASA Astrophysics Data System (ADS)

    Zhang, Qiuxiang; Lu, Rongwen; Wang, Benquan; Messinger, Jeffrey D.; Curcio, Christine A.; Yao, Xincheng

    2015-04-01

    Transient intrinsic optical signal (IOS) changes have been observed in retinal photoreceptors, suggesting a unique biomarker for eye disease detection. However, clinical deployment of IOS imaging is challenging due to unclear IOS sources and limited signal-to-noise ratios (SNRs). Here, by developing high spatiotemporal resolution optical coherence tomography (OCT) and applying an adaptive algorithm for IOS processing, we were able to record robust IOSs from single-pass measurements. Transient IOSs, which might reflect an early stage of light phototransduction, are consistently observed in the photoreceptor outer segment almost immediately (<4 ms) after retinal stimulation. Comparative studies of dark- and light-adapted retinas have demonstrated the feasibility of functional OCT mapping of rod and cone photoreceptors, promising a new method for early disease detection and improved treatment of diseases such as age-related macular degeneration (AMD) and other eye diseases that can cause photoreceptor damage.

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

    NASA Astrophysics Data System (ADS)

    Du, Congwu; Pan, Yingtian

    2014-03-01

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

  10. Brain basis of early parent–infant interactions: psychology, physiology, and in vivo functional neuroimaging studies

    PubMed Central

    Swain, James E.; Lorberbaum, Jeffrey P.; Kose, Samet; Strathearn, Lane

    2015-01-01

    Parenting behavior critically shapes human infants’ current and future behavior. The parent–infant relationship provides infants with their first social experiences, forming templates of what they can expect from others and how to best meet others’ expectations. In this review, we focus on the neurobiology of parenting behavior, including our own functional magnetic resonance imaging (fMRI) brain imaging experiments of parents. We begin with a discussion of background, perspectives and caveats for considering the neurobiology of parent–infant relationships. Then, we discuss aspects of the psychology of parenting that are significantly motivating some of the more basic neuroscience research. Following that, we discuss some of the neurohormones that are important for the regulation of social bonding, and the dysregulation of parenting with cocaine abuse. Then, we review the brain circuitry underlying parenting, proceeding from relevant rodent and nonhuman primate research to human work. Finally, we focus on a study-by-study review of functional neuroimaging studies in humans. Taken together, this research suggests that networks of highly conserved hypothalamic–midbrain–limbic–paralimbic–cortical circuits act in concert to support aspects of parent response to infants, including the emotion, attention, motivation, empathy, decision-making and other thinking that are required to navigate the complexities of parenting. Specifically, infant stimuli activate basal forebrain regions, which regulate brain circuits that handle specific nurturing and caregiving responses and activate the brain’s more general circuitry for handling emotions, motivation, attention, and empathy – all of which are crucial for effective parenting. We argue that an integrated understanding of the brain basis of parenting has profound implications for mental health. PMID:17355399

  11. Mechanism of platelet functional changes and effects of anti-platelet agents on in vivo hemostasis under different gravity conditions.

    PubMed

    Li, Suping; Shi, Quanwei; Liu, Guanglei; Zhang, Weilin; Wang, Zhicheng; Wang, Yuedan; Dai, Kesheng

    2010-05-01

    Serious thrombotic and hemorrhagic problems or even fatalities evoked by either microgravity or hypergravity occur commonly in the world. We recently reported that platelet functions are inhibited in microgravity environments and activated under high-G conditions, which reveals the pathogenesis for gravity change-related hemorrhagic and thrombotic diseases. However, the mechanisms of platelet functional variations under different gravity conditions remain unclear. In this study we show that the amount of filamin A coimmunoprecipitated with GPIbalpha was enhanced in platelets exposed to modeled microgravity and, in contrast, was reduced in 8 G-exposed platelets. Hypergravity induced actin filament formation and redistribution, whereas actin filaments were reduced in platelets treated with modeled microgravity. Furthermore, intracellular Ca2+ levels were elevated by hypergravity. Pretreatment of platelets with the cell-permeable Ca2+ chelator BAPTA-AM had no effect on cytoskeleton reorganization induced by hypergravity but significantly reduced platelet aggregation induced by ristocetin/hypergravity. Two anti-platelet agents, aspirin and tirofiban, effectively reversed the shortened tail bleeding time and reduced the death rate of mice exposed to hypergravity. Furthermore, the increased P-selectin surface expression was obviously reduced in platelets from mice treated with aspirin/hypergravity compared with those from mice treated with hypergravity alone. These data suggest that the actin cytoskeleton reorganization and intracellular Ca2+ level play key roles in the regulation of platelet functions in different gravitational environments. The results with anti-platelet agents not only further confirm the activation of platelets in vivo but also suggest a therapeutic potential for hypergravity-induced thrombotic diseases. PMID:20133435

  12. Immunologic effector mechanisms of a standardized mistletoe extract on the function of human monocytes and lymphocytes in vitro, ex vivo, and in vivo.

    PubMed

    Heinzerling, Lucie; von Baehr, Volker; Liebenthal, Christa; von Baehr, Rüdiger; Volk, Hans-Dieter

    2006-07-01

    Even though mistletoe extracts have been in clinical use for centuries their exact mode of action is still unknown. Currently, the application scheme for registered preparations is a dose-escalating scheme to thus reduce side effects. In this study, healthy controls and patients were evaluated for their immunologic response to treatment with a standardized mistletoe extract (Iscador). It shows a strong effect as adjuvant that induces TNF-alpha and IL-12, which was partly mediated via CD14. Desensitization of the TNF-alpha response could be shown after repeated application in vitro and in vivo. Furthermore, Iscador induces a specific lymphocyte sensitization upon multiple injections and production of IgG1- and IgG3 -mistletoe antibodies. Remarkably, a systemic bystander effect (heterologous immunity against other recall antigens) was observed after long-term treatment. In conclusion, dose-escalation reduces the monocyte-related clinical side effects. A T-lymphocyte sensitization stimulates mainly a specific Th1 response. The most interesting clinical long-term effect is the bystander stimulation of various memory T cells that might mediate in vivo antitumor and antiinfectious T-cell response under mistletoe-extract immunization. PMID:16705487

  13. Effect of Tomato Industrial Processing (Different Hybrids, Paste, and Pomace) on Inhibition of Platelet Function In Vitro, Ex Vivo, and In Vivo

    PubMed Central

    Rodríguez-Azúa, Rosio; Treuer, Adriana; Moore-Carrasco, Rodrigo; Cortacáns, Daniel; Gutiérrez, Margarita; Astudillo, Luis; Fuentes, Eduardo

    2014-01-01

    Abstract Cardiovascular disease (CVD) is the leading cause of death worldwide. Healthy eating is among its safeguards, especially the daily intake of fruits and vegetables. In this context it has been shown that tomato (Solanum lycopersicum) presents antiplatelet activity. In the present study, we evaluated in vitro antiplatelet activity of fresh hybrid tomato process (nine hybrids: Apt 410, H 9888, Bos 8066, Sun 6366, AB3, HMX 7883, H 9665, H 7709, and H 9997), paste and its by-product of industrial processes (pomace). We assessed antiplatelet activity ex vivo and bleeding time in rats that ingested 0.1 and 1.0?g/kg of pomace each day. In studies in vitro, no significant differences in antiplatelet activity was observed in fresh tomato hybrids. Furthermore, the agro-industrial process did not affect the antiplatelet activity of paste and pomace. Likewise, pomace intake of 1.0?g/kg per day prolonged bleeding time and reduced ex vivo platelet aggregation in rats. The data obtained indicate that tomato has one or more compounds that caused antiplatelet activity. Regular consumption of tomato and its industrial derivatives could be part of a CVD prevention regimen. PMID:24325459

  14. Selenite-Releasing Bone Mineral Nanoparticles Retard Bone Tumor Growth and Improve Healthy Tissue Functions In Vivo.

    PubMed

    Wang, Yanhua; Hao, Hang; Liu, Haoming; Wang, Yifan; Li, Yan; Yang, Gaojie; Ma, Jun; Mao, Chuanbin; Zhang, Shengmin

    2015-08-26

    Selenite-doped bone mineral nanoparticles can retard the growth of osteosarcoma in a nude mice model, through sustained release of selenite ions. The selenite ions released from the nanoparticles through a degradation-mediated fashion inhibit tumor metastasis. Blood routine analysis indicates that selenite ions can also improve the functions of liver, kidney, and heart. PMID:26101804

  15. In vivo alterations in skeletal muscle form and function after disuse atrophy.

    PubMed

    Clark, Brian C

    2009-10-01

    Prolonged reductions in muscle activity and mechanical loading (e.g., bed rest, cast immobilization) result in alterations in skeletal muscle form and function. The purpose of this review article was to synthesize recent findings from several studies on the dramatic effects of disuse on skeletal muscle morphology and muscle performance in humans. Specifically, the following are discussed: 1) how the antigravity muscles are most susceptible to atrophy and how the degree of atrophy varies between muscle groups; 2) how disuse alters muscle composition by increasing intermuscular adipose tissue; 3) the influence of different disuse models on regulating the loss of muscle mass and strength, with immobilization causing greater reductions than bed rest and limb suspension do; 4) the observation that disuse decreases strength to a greater extent than muscle mass and the role of adaptations in both neural and contractile properties that influences this excessive loss of strength; 5) the equivocal findings on the effect of disuse on muscle fatigue resistance; and 6) the reduction in motor control after prolonged disuse. Lastly, emerging data warranting further inquiry into the modulating role of biological sex on disuse-induced adaptations are also discussed. PMID:19727027

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

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

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

  17. Structure-function analysis of the histidine permease and comparison with cystic fibrosis mutations.

    PubMed

    Shyamala, V; Baichwal, V; Beall, E; Ames, G F

    1991-10-01

    Traffic ATPases constitute a superfamily of transporters that include prokaryotic permeases and medically important eukaryotic proteins, such as the multidrug resistance P-glycoprotein and the cystic fibrosis gene product. We present a structure-function analysis of a member of this superfamily, the prokaryotic histidine permease, using mutations generated both in vitro and in vivo, and assaying several biochemical functions. The analysis supports a previously predicted structural model and allows the assignment of specific functions to several predicted structural features. Mutations in the secondary structure features which form the nucleotide-binding pocket in general cause the loss of ATP binding activity. Mutations in the helical domain retain ATP binding activity. Several mutations have been identified which may affect the signaling mechanism between ATP hydrolysis and membrane translocation. We relate our findings to those emerging from the recent biochemical and genetic analyses of cystic fibrosis mutations. PMID:1717452

  18. A Dosimetry Study of Deuterium-Deuterium Neutron Generator-based In Vivo Neutron Activation Analysis.

    PubMed

    Sowers, Daniel; Liu, Yingzi; Mostafaei, Farshad; Blake, Scott; Nie, Linda H

    2015-12-01

    A neutron irradiation cavity for in vivo neutron activation analysis (IVNAA) to detect manganese, aluminum, and other potentially toxic elements in human hand bone has been designed and its dosimetric specifications measured. The neutron source is a customized deuterium-deuterium neutron generator that produces neutrons at 2.45 MeV by the fusion reaction H(d, n)He at a calculated flux of 7 × 10 ± 30% s. A moderator/reflector/shielding [5 cm high density polyethylene (HDPE), 5.3 cm graphite and 5.7 cm borated (HDPE)] assembly has been designed and built to maximize the thermal neutron flux inside the hand irradiation cavity and to reduce the extremity dose and effective dose to the human subject. Lead sheets are used to attenuate bremsstrahlung x rays and activation gammas. A Monte Carlo simulation (MCNP6) was used to model the system and calculate extremity dose. The extremity dose was measured with neutron and photon sensitive film badges and Fuji electronic pocket dosimeters (EPD). The neutron ambient dose outside the shielding was measured by Fuji NSN3, and the photon dose was measured by a Bicron MicroREM scintillator. Neutron extremity dose was calculated to be 32.3 mSv using MCNP6 simulations given a 10-min IVNAA measurement of manganese. Measurements by EPD and film badge indicate hand dose to be 31.7 ± 0.8 mSv for neutrons and 4.2 ± 0.2 mSv for photons for 10 min; whole body effective dose was calculated conservatively to be 0.052 mSv. Experimental values closely match values obtained from MCNP6 simulations. These are acceptable doses to apply the technology for a manganese toxicity study in a human population. PMID:26509624

  19. In vivo and in vitro analyses of amygdalar function reveal a role for copper

    PubMed Central

    Gaier, E. D.; Rodriguiz, R. M.; Zhou, J.; Ralle, M.; Wetsel, W. C.; Eipper, B. A.

    2014-01-01

    Mice with a single copy of the peptide amidating monooxygenase (Pam) gene (PAM+/?) are impaired in contextual and cued fear conditioning. These abnormalities coincide with deficient long-term potentiation (LTP) at excitatory thalamic afferent synapses onto pyramidal neurons in the lateral amygdala. Slice recordings from PAM+/? mice identified an increase in GABAergic tone (Gaier ED, Rodriguiz RM, Ma XM, Sivaramakrishnan S, Bousquet-Moore D, Wetsel WC, Eipper BA, Mains RE. J Neurosci 30: 13656–13669, 2010). Biochemical data indicate a tissue-specific deficit in Cu content in the amygdala; amygdalar expression of Atox-1 and Atp7a, essential for transport of Cu into the secretory pathway, is reduced in PAM+/? mice. When PAM+/? mice were fed a diet supplemented with Cu, the impairments in fear conditioning were reversed, and LTP was normalized in amygdala slice recordings. A role for endogenous Cu in amygdalar LTP was established by the inhibitory effect of a brief incubation of wild-type slices with bathocuproine disulfonate, a highly selective, cell-impermeant Cu chelator. Interestingly, bath-applied CuSO4 had no effect on excitatory currents but reversibly potentiated the disynaptic inhibitory current. Bath-applied CuSO4 was sufficient to potentiate wild-type amygdala afferent synapses. The ability of dietary Cu to affect signaling in pathways that govern fear-based behaviors supports an essential physiological role for Cu in amygdalar function at both the synaptic and behavioral levels. This work is relevant to neurological and psychiatric disorders in which disturbed Cu homeostasis could contribute to altered synaptic transmission, including Wilson's, Menkes, Alzheimer's, and prion-related diseases. PMID:24554785

  20. In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates.

    PubMed

    Sabino, Fabio; Hermes, Olivia; Egli, Fabian E; Kockmann, Tobias; Schlage, Pascal; Croizat, Pierre; Kizhakkedathu, Jayachandran N; Smola, Hans; auf dem Keller, Ulrich

    2015-02-01

    Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ?450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease-substrate relations in the healing skin wound in vivo. The data have been deposited to the ProteomeXchange Consortium with identifier PXD001198. PMID:25516628

  1. Function of the C-terminal Domain of the DEAD-Box Protein Mss116p Analyzed In Vivo and In Vitro

    PubMed Central

    Mohr, Georg; Del Campo, Mark; Mohr, Sabine; Yang, Quansheng; Jia, Huijue; Jankowsky, Eckhard; Lambowitz, Alan M.

    2008-01-01

    The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are general RNA chaperones that function in splicing mitochondrial group I and group II introns and in translational activation. Both proteins consist of a conserved ATP-dependent RNA helicase core region linked to N- and C-terminal domains, the latter with a basic tail similar to many other DEAD-box proteins. In CYT-19, this basic tail was shown to contribute to non-specific RNA binding that helps tether the core helicase region to structured RNA substrates. Here, multiple sequence alignments and secondary structure predictions indicate that CYT-19 and Mss116p belong to distinct subgroups of DEAD-box proteins, whose C-terminal domains have a defining extended ?-helical region preceding the basic tail. We find that mutations or C-terminal truncations in the predicted ?-helical region of Mss116p strongly inhibit RNA-dependent ATPase activity, leading to loss of function in both translational activation and RNA splicing. These findings suggest that the ?-helical region may stabilize and/or regulate the activity of the RNA helicase core. By contrast, a truncation that removes only the basic tail leaves high RNA-dependent ATPase activity and causes only a modest reduction in translation and RNA splicing efficiency in vivo and in vitro. Biochemical analysis shows that deletion of the basic tail leads to weaker non-specific binding of group I and group II intron RNAs, and surprisingly, also impairs RNA-unwinding at saturating protein concentrations and nucleotide-dependent tight binding of single-stranded RNAs by the RNA helicase core. Together, our results indicate that the two subregions of Mss116p’s C-terminal domain act in different ways to support and modulate activities of the core helicase region, whose RNA-unwinding activity is critical for both the translation and RNA splicing functions. PMID:18096186

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

    PubMed

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

    2012-01-01

    Tardigrades are microscopic aquatic animals with remarkable abilities to withstand harsh physical conditions such as dehydration or exposure to harmful highly energetic radiation. The mechanisms responsible for such robustness are presently little known, but protection against oxidative stresses is thought to play a role. Despite the fact that many tardigrade species are variously pigmented, scarce information is available about this characteristic. By applying Raman micro-spectroscopy on living specimens, pigments in the tardigrade Echiniscus blumi are identified as carotenoids, and their distribution within the animal body is visualized. The dietary origin of these pigments is demonstrated, as well as their presence in the eggs and in eye-spots of these animals, together with their absence in the outer layer of the animal (i.e., cuticle and epidermis). Using in-vivo semi-quantitative Raman micro-spectroscopy, a decrease in carotenoid content is detected after inducing oxidative stress, demonstrating that this approach can be used for studying the role of carotenoids in oxidative stress-related processes in tardigrades. This approach could be thus used in further investigations to test several hypotheses concerning the function of these carotenoids in tardigrades as photo-protective pigments against ionizing radiations or as antioxidants defending these organisms against the oxidative stress occurring during desiccation processes. PMID:23185564

  3. Myozap, a novel intercalated disc protein, activates SRF-dependent signaling and is required to maintain cardiac function in vivo

    PubMed Central

    Seeger, Thalia S.; Frank, Derk; Rohr, Claudia; Will, Rainer; Just, Steffen; Grund, Christine; Lyon, Robert; Lüdde, Mark; Koegl, Manfred; Sheikh, Farah; Rottbauer, Wolfgang; Franke, Werner W.; Katus, Hugo A.; Olson, Eric N.; Frey, Norbert

    2010-01-01

    Rationale The intercalated disc (ID) is a highly specialized cell-cell contact structure that ensures mechanical and electrical coupling of contracting cardiomyocytes. Recently, the ID has been recognized to be a hot spot of cardiac disease, in particular inherited cardiomyopathy. Objective Given its complex structure and function we hypothesized that important molecular constituents of the ID still remain unknown. Methods Using a bioinformatic screen, we discovered and cloned a previously uncharacterized 54 kDa cardiac protein which we termed Myozap (Myocardium-enriched ZO-associated protein). Results Myozap is strongly expressed in the heart and lung. In cardiac tissue it localized to the ID and directly binds to desmoplakin and ZO-1. In a yeast-two hybrid screen for additional binding partners of Myozap we identified myosin phosphatase-RhoA interacting protein (MRIP), a negative regulator of Rho activity. Myozap, in turn, strongly activates SRF-dependent transcription through its ERM (Ezrin/radixin/moesin)-like domain in a Rho-dependent fashion. Finally, in vivo knockdown of the Myozap orthologue in zebrafish led to severe contractile dysfunction and cardiomyopathy. Conclusions Taken together, these findings reveal Myozap as a previously unrecognized component of a Rho-dependent signaling pathway that links the intercalated disc to cardiac gene regulation. Moreover, its subcellular localization and the observation of a severe cardiac phenotype in zebrafish, implicate Myozap in the pathogenesis of cardiomyopathy. PMID:20093627

  4. Functional analysis of problem behavior: a review.

    PubMed Central

    Hanley, Gregory P; Iwata, Brian A; McCord, Brandon E

    2003-01-01

    Functional analysis methodology focuses on the identification of variables that influence the occurrence of problem behavior and has become a hallmark of contemporary approaches to behavioral assessment. In light of the widespread use of pretreatment functional analyses in articles published in this and other journals, we reviewed the literature in an attempt to identify best practices and directions for future research. Studies included in the present review were those in which (a) a pretreatment assessment based on (b) direct observation and measurement of (c) problem behavior was conducted under (d) at least two conditions involving manipulation of an environmental variable in an attempt (e) to demonstrate a relation between the environmental event and behavior. Studies that met the criteria for inclusion were quantified and critically evaluated along a number of dimensions related to subject and setting characteristics, parametric and qualitative characteristics of the methodology, types of assessment conditions, experimental designs, topographies of problem behaviors, and the manner in which data were displayed and analyzed. PMID:12858983

  5. Human milk metagenome: a functional capacity analysis

    PubMed Central

    2013-01-01

    Background Human milk contains a diverse population of bacteria that likely influences colonization of the infant gastrointestinal tract. Recent studies, however, have been limited to characterization of this microbial community by 16S rRNA analysis. In the present study, a metagenomic approach using Illumina sequencing of a pooled milk sample (ten donors) was employed to determine the genera of bacteria and the types of bacterial open reading frames in human milk that may influence bacterial establishment and stability in this primal food matrix. The human milk metagenome was also compared to that of breast-fed and formula-fed infants’ feces (n?=?5, each) and mothers’ feces (n?=?3) at the phylum level and at a functional level using open reading frame abundance. Additionally, immune-modulatory bacterial-DNA motifs were also searched for within human milk. Results The bacterial community in human milk contained over 360 prokaryotic genera, with sequences aligning predominantly to the phyla of Proteobacteria (65%) and Firmicutes (34%), and the genera of Pseudomonas (61.1%), Staphylococcus (33.4%) and Streptococcus (0.5%). From assembled human milk-derived contigs, 30,128 open reading frames were annotated and assigned to functional categories. When compared to the metagenome of infants’ and mothers’ feces, the human milk metagenome was less diverse at the phylum level, and contained more open reading frames associated with nitrogen metabolism, membrane transport and stress response (P?functionality of the human milk metagenome are warranted. PMID:23705844

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

    PubMed Central

    2012-01-01

    Background Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA) and its chaperone (LipB) from a screened strain named AB which belongs to Pseudomonas aeruginosa were overexpressed in E. coli with two dual expression plasmid systems to enhance the production of the active lipase LipA without in vitro refolding process. Results In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as P. aeruginosa on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate the gene lipA (936 bp) and lipase specific foldase gene lipB (1023 bp). One single expression plasmid system E. coli BL21/pET28a-lipAB and two dual expression plasmid systems E. coli BL21/pETDuet-lipA-lipB and E. coli BL21/pACYCDuet-lipA-lipB were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by E. coli BL21/pET28a-lipAB and E. coli BL21/pETDuet-lipA-lipB were 1300 U/L and 3200 U/L, respectively, while the activity of the lipase expressed by E. coli BL21/pACYCDuet-lipA-lipB was up to 8500 U/L. The lipase LipA had an optimal temperature of 30°C and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol, respectively. Conclusions The effect of different plasmid system on the active LipA expression was significantly different. pACYCDuet-lipA-lipB was more suitable for the expression of active LipA than pET28a-lipAB and pETDuet-lipA-lipB. The LipA showed obvious esterification activity and thus had potential biocatalytic applications. The expression method reported here can give reference for the expression of those enzymes that require chaperones. PMID:22950599

  7. A novel single walled carbon nanotube (SWCNT) functionalization agent facilitating in vivo combined chemo/thermo therapy

    NASA Astrophysics Data System (ADS)

    Zhang, Liwen; Rong, Pengfei; Chen, Minglong; Gao, Shi; Zhu, Lei

    2015-10-01

    Carbon nanotubes (CNTs) have shown intriguing applications in biotechnological and biomedical fields due to their unique shape and properties. However, the fact that unmodified CNTs are prone to aggregation, stunts CNTs applications under physiological conditions. In this research, we found that as little as 1/5th the single walled carbon nanotube (SWCNT) weight of Evans Blue (EB) is capable of dispersing SWCNT as well as facilitating SWCNT functionalization. In view of the binding between EB and albumin, the yielding product (SWCNT/EB) demonstrated extreme stability for weeks under physiological conditions and it can be endowed with a therapeutic ability by simply mixing SWCNT/EB with an albumin based drug. Specifically, the formed SWCNT/EB/albumin/PTX nanocomplex exhibits strong near-infrared (NIR) absorbance, and can serve as an agent for chemo/thermal therapeutic purposes. Our in vivo result reveals that SWCNT/EB/albumin/PTX after being administered into the MDA-MB-435 tumor would effectively ablate the tumor by chemo and photothermal therapy. Such a combined treatment strategy provides remarkable therapeutic outcomes in restraining tumor growth compared to chemo or photothermal therapy alone. Overall, our strategy of dispersing SWCNTs by EB can be used as a platform for carrying other drugs or functional genes with the aid of albumin to treat diseases. The present study opens new opportunities in surface modification of SWCNTs for future clinical disease treatment.Carbon nanotubes (CNTs) have shown intriguing applications in biotechnological and biomedical fields due to their unique shape and properties. However, the fact that unmodified CNTs are prone to aggregation, stunts CNTs applications under physiological conditions. In this research, we found that as little as 1/5th the single walled carbon nanotube (SWCNT) weight of Evans Blue (EB) is capable of dispersing SWCNT as well as facilitating SWCNT functionalization. In view of the binding between EB and albumin, the yielding product (SWCNT/EB) demonstrated extreme stability for weeks under physiological conditions and it can be endowed with a therapeutic ability by simply mixing SWCNT/EB with an albumin based drug. Specifically, the formed SWCNT/EB/albumin/PTX nanocomplex exhibits strong near-infrared (NIR) absorbance, and can serve as an agent for chemo/thermal therapeutic purposes. Our in vivo result reveals that SWCNT/EB/albumin/PTX after being administered into the MDA-MB-435 tumor would effectively ablate the tumor by chemo and photothermal therapy. Such a combined treatment strategy provides remarkable therapeutic outcomes in restraining tumor growth compared to chemo or photothermal therapy alone. Overall, our strategy of dispersing SWCNTs by EB can be used as a platform for carrying other drugs or functional genes with the aid of albumin to treat diseases. The present study opens new opportunities in surface modification of SWCNTs for future clinical disease treatment. Electronic supplementary information (ESI) available: Characterization of EB dispersed SWCNT; chemical structures of dyes applied for SWCNT dispersion; spectrum of EB/albumin; PTX loading efficiency onto albumin at different ratios. See DOI: 10.1039/c5nr03752b

  8. Parametric Cost Analysis: A Design Function

    NASA Technical Reports Server (NTRS)

    Dean, Edwin B.

    1989-01-01

    Parametric cost analysis uses equations to map measurable system attributes into cost. The measures of the system attributes are called metrics. The equations are called cost estimating relationships (CER's), and are obtained by the analysis of cost and technical metric data of products analogous to those to be estimated. Examples of system metrics include mass, power, failure_rate, mean_time_to_repair, energy _consumed, payload_to_orbit, pointing_accuracy, manufacturing_complexity, number_of_fasteners, and percent_of_electronics_weight. The basic assumption is that a measurable relationship exists between system attributes and the cost of the system. If a function exists, the attributes are cost drivers. Candidates for metrics include system requirement metrics and engineering process metrics. Requirements are constraints on the engineering process. From optimization theory we know that any active constraint generates cost by not permitting full optimization of the objective. Thus, requirements are cost drivers. Engineering processes reflect a projection of the requirements onto the corporate culture, engineering technology, and system technology. Engineering processes are an indirect measure of the requirements and, hence, are cost drivers.

  9. In vitro and in vivo evaluation of the effects of piperine on P-gp function and expression.

    PubMed

    Han, Yi; Chin Tan, Theresa May; Lim, Lee-Yong

    2008-08-01

    Piperine, a major component of black pepper, is used as spice and nutrient enhancer. The purpose of the present study was to evaluate the effects of acute and prolonged piperine exposure on cellular P-gp expression and function in vitro and in vivo. Piperine at concentrations ranging from 10 to 100 microM, determined by MTT assay to be non-cytotoxic, was observed to inhibit P-gp mediated efflux transport of [(3)H]-digoxin across L-MDR1 and Caco-2 cell monolayers. The acute inhibitory effect was dependent on piperine concentration, with abolishment of [(3)H]-digoxin polarized transport attained at 50 microM of piperine. In contrast, prolonged (48 and 72 h) co-incubation of Caco-2 cell monolayers with piperine (50 and 100 microM) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by a higher degree of [(3)H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. Peroral administration of piperine at the dose of 112 microg/kg body weight/day to male Wistar rats for 14 consecutive days also led to increased intestinal P-gp levels. However, there was a concomitant reduction in the rodent liver P-gp although the kidney P-gp level was unaffected. Our data suggest that caution should be exercised when piperine is to be co-administered with drugs that are P-gp substrates, particularly for patients whose diet relies heavily on pepper. PMID:18417181

  10. Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells

    PubMed Central

    2013-01-01

    Background Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay. Results Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation. In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system. Conclusions The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion. PMID:24168020

  11. In vitro and in vivo evaluation of the effects of piperine on P-gp function and expression

    SciTech Connect

    Han Yi; Chin Tan, Theresa May; Lim, Lee-Yong

    2008-08-01

    Piperine, a major component of black pepper, is used as spice and nutrient enhancer. The purpose of the present study was to evaluate the effects of acute and prolonged piperine exposure on cellular P-gp expression and function in vitro and in vivo. Piperine at concentrations ranging from 10 to 100 {mu}M, determined by MTT assay to be non-cytotoxic, was observed to inhibit P-gp mediated efflux transport of [{sup 3}H]-digoxin across L-MDR1 and Caco-2 cell monolayers. The acute inhibitory effect was dependent on piperine concentration, with abolishment of [{sup 3}H]-digoxin polarized transport attained at 50 {mu}M of piperine. In contrast, prolonged (48 and 72 h) co-incubation of Caco-2 cell monolayers with piperine (50 and 100 {mu}M) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by a higher degree of [{sup 3}H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. Peroral administration of piperine at the dose of 112 {mu}g/kg body weight/day to male Wistar rats for 14 consecutive days also led to increased intestinal P-gp levels. However, there was a concomitant reduction in the rodent liver P-gp although the kidney P-gp level was unaffected. Our data suggest that caution should be exercised when piperine is to be co-administered with drugs that are P-gp substrates, particularly for patients whose diet relies heavily on pepper.

  12. Potential electron mediators to extract electron energies of RBC glycolysis for prolonged in vivo functional lifetime of hemoglobin vesicles.

    PubMed

    Kettisen, Karin; Bülow, Leif; Sakai, Hiromi

    2015-04-15

    Developing a functional blood substitute as an alternative to donated blood for clinical use is believed to relieve present and future blood shortages, and to reduce the risks of infection and blood type mismatching. Hemoglobin vesicle (HbV) encapsulates a purified and concentrated human-derived Hb solution in a phospholipid vesicle (liposome). The in vivo safety and efficacy of HbV as a transfusion alternative have been clarified. Auto-oxidation of ferrous Hb in HbV gradually increases the level of ferric methemoglobin (metHb) and impairs the oxygen transport capabilities. The extension of the functional half-life of HbV has recently been proposed using an electron mediator, methylene blue (MB), which acts as a shuttle between red blood cells (RBC) and HbV. MB transfers electron energies of NAD(P)H, produced by RBC glycolysis, to metHb in HbV. Work presented here focuses on screening of 15 potential electron mediators, with appropriate redox potential and water solubility, for electron transfer from RBC to HbV. The results are assessed with regard to the chemical properties of the candidates. The compounds examined in this study were dimethyl methylene blue (DMB), methylene green, azure A, azure B, azure C, toluidine blue (TDB), thionin acetate, phenazine methosulfate, brilliant cresyl blue, cresyl violet, gallocyanine, toluylene blue, indigo carmine, indigotetrasulfonate, and MB. Six candidates were found to be unsuitable because of their insufficient diffusion across membranes, or overly high or nonexistent reactivity with relevant biomolecules. However, 9 displayed favorable metHb reduction. Among the suitable candidates, phenothiazines DMB and TDB exhibited effectiveness like MB did. In comparison to MB, they showed faster reduction by electron-donating NAD(P)H, coupled with showing a lower rate of reoxidation in the presence of molecular oxygen. Ascertaining the best electron mediator can provide a pathway for extending the lifetime and efficiency of potential blood substitutes. PMID:25734688

  13. Analysis of Single Locus Trajectories for Extracting In Vivo Chromatin Tethering Interactions

    PubMed Central

    Amitai, Assaf; Toulouze, Mathias; Dubrana, Karine; Holcman, David

    2015-01-01

    Is it possible to extract tethering forces applied on chromatin from the statistics of a single locus trajectories imaged in vivo? Chromatin fragments interact with many partners such as the nuclear membrane, other chromosomes or nuclear bodies, but the resulting forces cannot be directly measured in vivo. However, they impact chromatin dynamics and should be reflected in particular in the motion of a single locus. We present here a method based on polymer models and statistics of single trajectories to extract the force characteristics and in particular when they are generated by the gradient of a quadratic potential well. Using numerical simulations of a Rouse polymer and live cell imaging of the MAT-locus located on the yeast Saccharomyces cerevisiae chromosome III, we recover the amplitude and the distance between the observed and the interacting monomer. To conclude, the confined trajectories we observed in vivo reflect local interaction on chromatin. PMID:26317360

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

    SciTech Connect

    Fornace, Jr, A J

    2007-03-03

    Abstract for final report for project entitled â??A functional genomics approach using radiation-induced changes in gene expression to study low dose radiation effects in vitro and in vivoâ?ť which has been supported by the DOE Low Dose Radiation Research Program for approximately 7 years. This project has encompassed two sequential awards, ER62683 and then ER63308, in the Gene Response Section in the Center for Cancer Research at the National Cancer Institute. The project was temporarily suspended during the relocation of the Principal Investigatorâ??s laboratory to the Dept. of Genetics and Complex Diseases at Harvard School of Public Health at the end of 2004. Remaining support for the final year was transferred to this new site later in 2005 and was assigned the DOE Award Number ER64065. The major aims of this project have been 1) to characterize changes in gene expression in response to low-dose radiation responses; this includes responses in human cells lines, peripheral blood lymphocytes (PBL), and in vivo after human or murine exposures, as well as the effect of dose-rate on gene responses; 2) to characterize changes in gene expression that may be involved in bystander effects, such as may be mediated by cytokines and other intercellular signaling proteins; and 3) to characterize responses in transgenic mouse models with relevance to genomic stability. A variety of approaches have been used to study transcriptional events including microarray hybridization, quantitative single-probe hybridization which was developed in this laboratory, quantitative RT-PCR, and promoter microarray analysis using genomic regulatory motifs. Considering the frequent responsiveness of genes encoding cytokines and related signaling proteins that can affect cellular metabolism, initial efforts were initiated to study radiation responses at the metabolomic level and to correlate with radiation-responsive gene expression. Productivity includes twenty-four published and in press manuscripts, as well as a U.S. patent. There are several additional publications that will be submitted in 2007 that were supported in part by this program. These future publications include one manuscript on in vivo expression profiling analysis in mouse models, one manuscript on radiation responses in human cell lines, at least one on development of stress signatures in human cells, and three manuscripts on radiation metabolomics.

  15. Functional Analysis of Variance for Association Studies

    PubMed Central

    Vsevolozhskaya, Olga A.; Zaykin, Dmitri V.; Greenwood, Mark C.; Wei, Changshuai; Lu, Qing

    2014-01-01

    While progress has been made in identifying common genetic variants associated with human diseases, for most of common complex diseases, the identified genetic variants only account for a small proportion of heritability. Challenges remain in finding additional unknown genetic variants predisposing to complex diseases. With the advance in next-generation sequencing technologies, sequencing studies have become commonplace in genetic research. The ongoing exome-sequencing and whole-genome-sequencing studies generate a massive amount of sequencing variants and allow researchers to comprehensively investigate their role in human diseases. The discovery of new disease-associated variants can be enhanced by utilizing powerful and computationally efficient statistical methods. In this paper, we propose a functional analysis of variance (FANOVA) method for testing an association of sequence variants in a genomic region with a qualitative trait. The FANOVA has a number of advantages: (1) it tests for a joint effect of gene variants, including both common and rare; (2) it fully utilizes linkage disequilibrium and genetic position information; and (3) allows for either protective or risk-increasing causal variants. Through simulations, we show that FANOVA outperform two popularly used methods – SKAT and a previously proposed method based on functional linear models (FLM), – especially if a sample size of a study is small and/or sequence variants have low to moderate effects. We conduct an empirical study by applying three methods (FANOVA, SKAT and FLM) to sequencing data from Dallas Heart Study. While SKAT and FLM respectively detected ANGPTL 4 and ANGPTL 3 associated with obesity, FANOVA was able to identify both genes associated with obesity. PMID:25244256

  16. Ex-vivo holographic microscopy and spectroscopic analysis of head and neck cancer

    NASA Astrophysics Data System (ADS)

    Holler, Stephen; Wurtz, Robert; Auyeung, Kelsey; Auyeung, Kris; Paspaley-Grbavac, Milan; Mulroe, Brigid; Sobrero, Maximiliano; Miles, Brett

    2015-03-01

    Optical probes to identify tumor margins in vivo would greatly reduce the time, effort and complexity in the surgical removal of malignant tissue in head and neck cancers. Current approaches involve visual microscopy of stained tissue samples to determine cancer margins, which results in the excision of excess of tissue to assure complete removal of the cancer. Such surgical procedures and follow-on chemotherapy can adversely affect the patient's recovery and subsequent quality of life. In order to reduce the complexity of the process and minimize adverse effects on the patient, we investigate ex vivo tissue samples (stained and unstained) using digital holographic microscopy in conjunction with spectroscopic analyses (reflectance and transmission spectroscopy) in order to determine label-free, optically identifiable characteristic features that may ultimately be used for in vivo processing of cancerous tissues. The tissue samples studied were squamous cell carcinomas and associated controls from patients of varying age, gender and race. Holographic microscopic imaging scans across both cancerous and non-cancerous tissue samples yielded amplitude and phase reconstructions that were correlated with spectral signatures. Though the holographic reconstructions and measured spectra indicate variations even among the same class of tissue, preliminary results indicate the existence of some discriminating features. Further analyses are presently underway to further this work and extract additional information from the imaging and spectral data that may prove useful for in vivo surgical identification.

  17. Ex vivo analysis identifies effective HIV-1 latency-reversing drug combinations.

    PubMed

    Laird, Gregory M; Bullen, C Korin; Rosenbloom, Daniel I S; Martin, Alyssa R; Hill, Alison L; Durand, Christine M; Siliciano, Janet D; Siliciano, Robert F

    2015-05-01

    Reversal of HIV-1 latency by small molecules is a potential cure strategy. This approach will likely require effective drug combinations to achieve high levels of latency reversal. Using resting CD4+ T cells (rCD4s) from infected individuals, we developed an experimental and theoretical framework to identify effective latency-reversing agent (LRA) combinations. Utilizing ex vivo assays for intracellular HIV-1 mRNA and virion production, we compared 2-drug combinations of leading candidate LRAs and identified multiple combinations that effectively reverse latency. We showed that protein kinase C agonists in combination with bromodomain inhibitor JQ1 or histone deacetylase inhibitors robustly induce HIV-1 transcription and virus production when directly compared with maximum reactivation by T cell activation. Using the Bliss independence model to quantitate combined drug effects, we demonstrated that these combinations synergize to induce HIV-1 transcription. This robust latency reversal occurred without release of proinflammatory cytokines by rCD4s. To extend the clinical utility of our findings, we applied a mathematical model that estimates in vivo changes in plasma HIV-1 RNA from ex vivo measurements of virus production. Our study reconciles diverse findings from previous studies, establishes a quantitative experimental approach to evaluate combinatorial LRA efficacy, and presents a model to predict in vivo responses to LRAs. PMID:25822022

  18. Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease and the pathogenic mechanisms of this important fish pathogen are not fully understood. Identifying bacterial components expressed in vivo may lead to a better understanding of pathogenesis and provide targets for va...

  19. Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease, and the pathogenic mechanisms of this important fish pathogen are not fully understood. Identifying bacterial genes of F. psychrophilum differentially expressed in vivo may lead to a better understanding of pathogen...

  20. Proteomic Analysis of Flavobacterium psychrophilum cultured In Vivo and In Iron-Limited Media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease and the pathogenic mechanisms of this important fish pathogen are not fully understood. Identifying bacterial components expressed in vivo may lead to a better understanding of pathogenesis and provide targets for va...

  1. 40 CFR 798.5385 - In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J. “Mammalian in vivo and vitro cytogenetics assays: Report of the Gene-Tox Program,” Mutation Research, 87:143-188 (1981). [50 FR 39397, Sept. 27, 1985, as amended at 52 FR...

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

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J. “Mammalian in vivo and vitro cytogenetics assays: Report of the Gene-Tox Program,” Mutation Research, 87:143-188 (1981). [50 FR 39397, Sept. 27, 1985, as amended at 52 FR...

  3. 40 CFR 798.5385 - In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J. “Mammalian in vivo and vitro cytogenetics assays: Report of the Gene-Tox Program,” Mutation Research, 87:143-188 (1981). [50 FR 39397, Sept. 27, 1985, as amended at 52 FR...

  4. 40 CFR 798.5385 - In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J. “Mammalian in vivo and vitro cytogenetics assays: Report of the Gene-Tox Program,” Mutation Research, 87:143-188 (1981). [50 FR 39397, Sept. 27, 1985, as amended at 52 FR...

  5. 40 CFR 798.5385 - In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J. “Mammalian in vivo and vitro cytogenetics assays: Report of the Gene-Tox Program,” Mutation Research, 87:143-188 (1981). [50 FR 39397, Sept. 27, 1985, as amended at 52 FR...

  6. Defective Mitochondrial Function In Vivo in Skeletal Muscle in Adults with Down’s Syndrome: A 31P-MRS Study

    PubMed Central

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

    2013-01-01

    Down’s syndrome (DS) is a developmental disorder associated with intellectual disability (ID). We have previously shown that people with DS engage in very low levels of exercise compared to people with ID not due to DS. Many aspects of the DS phenotype, such as dementia, low activity levels and poor muscle tone, are shared with disorders of mitochondrial origin, and mitochondrial dysfunction has been demonstrated in cultured DS tissue. We undertook a phosphorus magnetic resonance spectroscopy (31P-MRS) study in the quadriceps muscle of 14 people with DS and 11 non-DS ID controls to investigate the post-exercise resynthesis kinetics of phosphocreatine (PCr), which relies on mitochondrial respiratory function and yields a measure of muscle mitochondrial function in vivo. We found that the PCr recovery rate constant was significantly decreased in adults with DS compared to non-DS ID controls (1.7±0.1 min?1 vs 2.1±0.1 min?1 respectively) who were matched for physical activity levels, indicating that muscle mitochondrial function in vivo is impaired in DS. This is the first study to investigate mitochondrial function in vivo in DS using 31P-MRS. Our study is consistent with previous in vitro studies, supporting a theory of a global mitochondrial defect in DS. PMID:24391872

  7. Ex vivo lung perfusion to improve donor lung function and increase the number of organs available for transplantation

    PubMed Central

    Valenza, Franco; Rosso, Lorenzo; Coppola, Silvia; Froio, Sara; Palleschi, Alessandro; Tosi, Davide; Mendogni, Paolo; Salice, Valentina; Ruggeri, Giulia M; Fumagalli, Jacopo; Villa, Alessandro; Nosotti, Mario; Santambrogio, Luigi; Gattinoni, Luciano

    2014-01-01

    This paper describes the initial clinical experience of ex vivo lung perfusion (EVLP) at the Fondazione Ca’ Granda in Milan between January 2011 and May 2013. EVLP was considered if donor PaO2/FiO2 was below 300 mmHg or if lung function was doubtful. Donors with massive lung contusion, aspiration, purulent secretions, pneumonia, or sepsis were excluded. EVLP was run with a low-flow, open atrium and low hematocrit technique. Thirty-five lung transplants from brain death donors were performed, seven of which after EVLP. EVLP donors were older (54 ± 9 years vs. 40 ± 15 years, EVLP versus Standard, P < 0.05), had lower PaO2/FiO2 (264 ± 78 mmHg vs. 453 ± 119 mmHg, P < 0.05), and more chest X-ray abnormalities (P < 0.05). EVLP recipients were more often admitted to intensive care unit as urgent cases (57% vs. 18%, P = 0.05); lung allocation score at transplantation was higher (79 [40–84] vs. 39 [36–46], P < 0.05). After transplantation, primary graft dysfunction (PGD72 grade 3, 32% vs. 28%, EVLP versus Standard, P = 1), mortality at 30 days (0% vs. 0%, P = 1), and overall survival (71% vs. 86%, EVLP versus Standard P = 0.27) were not different between groups. EVLP enabled a 20% increase in available donor organs and resulted in successful transplants with lungs that would have otherwise been rejected (ClinicalTrials.gov number: NCT01967953). PMID:24628890

  8. Determination of dependencies among in vitro and in vivo properties of prepared mucoadhesive buccal films using multivariate data analysis.

    PubMed

    Vetchý, David; Landová, Hana; Gajdziok, Jan; Doležel, Petr; Dan?k, Zden?k; Štembírek, Jan

    2014-04-01

    Mucoadhesive films represent the most developed medical form of buccal application. Despite the intense focus on buccal film-based systems, there are no standardized methods for their evaluation, which limits the possibility of comparison of obtained data and evaluation of the significance of influence of formulation and process variables on properties of resulting films. The used principal component analysis, together with a partial least squares regression provided a unique insight into the effects of in vitro parameters of mucoadhesive buccal films on their in vivo properties and into interdependencies among the studied variables. In the present study eight various mucoadhesive buccal films based on mucoadhesive polymers (carmellose, polyethylene oxide) were prepared using a solvent casting method or a method of impregnation, respectively. An ethylcellulose or hydrophobic blend of white beeswax and white petrolatum were used as a backing layer. The addition of polyethylene oxide prolonged the in vivo film residence time (from 53.24±5.38-74.18±5.13 min to 71.05±3.15-98.12±1.75 min), and even more when combined with an ethylcellulose backing layer (98.12±1.75 min) and also improved the film's appearance. Tested non-woven textile shortened the in vivo film residence time (from 74.18±5.13-98.12±1.75 min to 53.24±5.38-81.00±8.47 min) and generally worsened the film's appearance. Mucoadhesive buccal films with a hydrophobic backing layer were associated with increased frequency of adverse effects. PMID:24333664

  9. Ferricyanide-backfilled cylindrical carbon fiber microelectrodes for in vivo analysis with high stability and low polarized potential.

    PubMed

    Zhong, Peipei; Yu, Ping; Wang, Kai; Hao, Jie; Fei, Junjie; Mao, Lanqun

    2015-11-01

    The development of stable and reproducible methods for in vivo electrochemical monitoring of neurochemicals is of great physiological importance. In this study, we demonstrate ferricyanide-filled cylindrical carbon fiber microelectrodes (CFEs) of high stability and low polarized potential for in vivo electrochemical analysis. We first studied the voltammetric behavior of cylindrical CFEs by using a model system consisting of two separated cells each containing potassium ferricyanide (K3Fe(CN)6) or potassium ferrocyanide (K4Fe(CN)6). We observed that E1/2 values of the system were dependent on the ratio of the lengths of the cylindrical CFEs and of the concentrations of the redox species on both poles. Based on this property, we prepared the ferricyanide-backfilled cylindrical CFEs, and found that this kind of electrode exhibits a more stable current response and a lower polarized potential than the CFEs backfilled with KCl or Ru(NH3)6Cl3. Animal experiments with the ferricyanide-backfilled cylindrical CFEs demonstrate that this kind of electrode could be used for in vivo monitoring of neurochemical release with a high stability under some physiological conditions. PMID:26378690

  10. An Integrated Tool for Functional Requirements Analysis, Function Allocation and Task Analysis

    SciTech Connect

    2015-03-02

    The software application is called ?HFE-Trace?. This is an integrated method and tool for the management of Human Factors Engineering analyses and related data. Its primary purpose is to support the coherent and consistent application of the nuclear industry?s best practices for human factors engineering work. The software is a custom Microsoft® Access® application. The application is used (in conjunction with other tools such as spreadsheets, checklists and normal documents where necessary) to collect data on the design of a new nuclear power plant from subject matter experts and other sources. This information is then used to identify potential system and functional breakdowns of the intended power plant design. This information is expanded by developing extensive descriptions of all functions, as well as system performance parameters, operating limits and constraints, and operational conditions. Once these have been verified, the human factors elements are added to each function, including intended operator role, function allocation considerations, prohibited actions, primary task categories, and primary work station. In addition, the application includes a computational method to assess a number of factors such as system and process complexity, workload, environmental conditions, procedures, regulations, etc.) that may shape operator performance. This is a unique methodology based upon principles described in NUREG/CR-3331 (?A methodology for allocating nuclear power plant control functions to human or automatic control?) and it results in a semi-quantified allocation of functions to three or more levels of automation for a conceptual automation system. The aggregate of all this information is then linked to the Task Analysis section of the application where the existing information on all operator functions is transformed into task information and ultimately into design requirements for Human-System Interfaces and Control Rooms. This final step includes assessment of methods to prevent potential operator errors.

  11. An Integrated Tool for Functional Requirements Analysis, Function Allocation and Task Analysis

    Energy Science and Technology Software Center (ESTSC)

    2015-03-02

    The software application is called ?HFE-Trace?. This is an integrated method and tool for the management of Human Factors Engineering analyses and related data. Its primary purpose is to support the coherent and consistent application of the nuclear industry?s best practices for human factors engineering work. The software is a custom Microsoft® Access® application. The application is used (in conjunction with other tools such as spreadsheets, checklists and normal documents where necessary) to collect datamore »on the design of a new nuclear power plant from subject matter experts and other sources. This information is then used to identify potential system and functional breakdowns of the intended power plant design. This information is expanded by developing extensive descriptions of all functions, as well as system performance parameters, operating limits and constraints, and operational conditions. Once these have been verified, the human factors elements are added to each function, including intended operator role, function allocation considerations, prohibited actions, primary task categories, and primary work station. In addition, the application includes a computational method to assess a number of factors such as system and process complexity, workload, environmental conditions, procedures, regulations, etc.) that may shape operator performance. This is a unique methodology based upon principles described in NUREG/CR-3331 (?A methodology for allocating nuclear power plant control functions to human or automatic control?) and it results in a semi-quantified allocation of functions to three or more levels of automation for a conceptual automation system. The aggregate of all this information is then linked to the Task Analysis section of the application where the existing information on all operator functions is transformed into task information and ultimately into design requirements for Human-System Interfaces and Control Rooms. This final step includes assessment of methods to prevent potential operator errors.« less

  12. Titanium implants with modified surfaces: meta-analysis of in vivo osteointegration.

    PubMed

    Gasik, Michael; Braem, Annabel; Chaudhari, Amol; Duyck, Joke; Vleugels, Jozef

    2015-04-01

    Titanium-based implants are widely used in modern clinical practice, but their "optimal" properties in terms of porosity and topology, roughness and hydrophilic parameters are being a subject of intensive discussions. Recent in vitro results have shown a possibility to optimize the surface of an implant with maximal repelling of bacteria (Staphylococcus aureus, Staphylococcus epidermidis) and improvement in human osteogenic and endothelial cell adhesion, proliferation and differentiation. In this work, these different grades titanium implants were tested in vivo using the same analytical methodology. In addition to material parameters, key histomorphometrical parameters such a regeneration area, bone adaptation area and bone-to-implant contact were determined after 2 and 4weeks of implantation in rabbit animal model. Porous implants have more clear differences than non-porous ones, with the best optimum values obtained on hydrothermally treated electrophoretically deposited titanium. These in vivo data correlate well with the optimal prediction made by in vitro tests. PMID:25686935

  13. Status Epilepticus Induced Spontaneous Dentate Gyrus Spikes: In Vivo Current Source Density Analysis

    PubMed Central

    Flynn, Sean P.; Barrier, Sylvain; Scott, Rod C.; Lenck- Santini, Pierre-Pascal; Holmes, Gregory L.

    2015-01-01

    The dentate gyrus is considered to function as an inhibitory gate limiting excitatory input to the hippocampus. Following status epilepticus (SE), this gating function is reduced and granule cells become hyper-excitable. Dentate spikes (DS) are large amplitude potentials observed in the dentate gyrus (DG) of normal animals. DS are associated with membrane depolarization of granule cells, increased activity of hilar interneurons and suppression of CA3 and CA1 pyramidal cell firing. Therefore, DS could act as an anti-excitatory mechanism. Because of the altered gating function of the dentate gyrus following SE, we sought to investigate how DS are affected following pilocarpine-induced SE. Two weeks following lithium-pilocarpine SE induction, hippocampal EEG was recorded in male Sprague-Dawley rats with 16-channel silicon probes under urethane anesthesia. Probes were placed dorso-ventrally to encompass either CA1-CA3 or CA1-DG layers. Large amplitude spikes were detected from EEG recordings and subject to current source density analysis. Probe placement was verified histologically to evaluate the anatomical localization of current sinks and the origin of DS. In 9 of 11 pilocarpine-treated animals and two controls, DS were confirmed with large current sinks in the molecular layer of the dentate gyrus. DS frequency was significantly increased in pilocarpine-treated animals compared to controls. Additionally, in pilocarpine-treated animals, DS displayed current sinks in the outer, middle and/or inner molecular layers. However, there was no difference in the frequency of events when comparing between layers. This suggests that following SE, DS can be generated by input from medial and lateral entorhinal cortex, or within the dentate gyrus. DS were associated with an increase in multiunit activity in the granule cell layer, but no change in CA1. These results suggest that following SE there is an increase in DS activity, potentially arising from hyperexcitability along the hippocampal-entorhinal pathway or within the dentate gyrus itself. PMID:26148195

  14. Analysis of ECs and related compounds in plasma: artifactual isomerization and ex vivo enzymatic generation of 2-MGs[S

    PubMed Central

    Pastor, Antoni; Farré, Magí; Fitó, Montserrat; Fernandez-Aranda, Fernando; de la Torre, Rafael

    2014-01-01

    The analysis of peripheral endocannabinoids (ECs) is a good biomarker of the EC system. Their concentrations, from clinical studies, strongly depend on sample collection and time processing conditions taking place in clinical and laboratory settings. The analysis of 2-monoacylglycerols (MGs) (i.e., 2-arachidonoylglycerol or 2-oleoylglycerol) is a particularly challenging issue because of their ex vivo formation and chemical isomerization that occur after blood sample collection. We provide evidence that their ex vivo formation can be minimized by adding Orlistat, an enzymatic lipase inhibitor, to plasma. Taking into consideration the low cost of Orlistat, we recommend its addition to plasma collecting tubes while maintaining sample cold chain until storage. We have validated a method for the determination of the EC profile of a range of MGs and N-acylethanolamides in plasma that preserves the original isomer ratio of MGs. Nevertheless, the chemical isomerization of 2-MGs can only be avoided by an immediate processing and analysis of samples due to their instability during conservation. We believe that this new methodology can aid in the harmonization of the measurement of ECs and related compounds in clinical samples. PMID:24610889

  15. Effect of Perinatal secondhand tobacco smoke exposure on in vivo and intrinsic airway structure/function in non-human primates

    SciTech Connect

    Joad, Jesse P. Kott, Kayleen S.; Bric, John M.; Peake, Janice L.; Pinkerton, Kent E.

    2009-02-01

    Infants exposed to second hand smoke (SHS) experience more problems with wheezing. This study was designed to determine if perinatal SHS exposure increases intrinsic and/or in vivo airway responsiveness to methacholine and whether potential structural/cellular alterations in the airway might explain the change in responsiveness. Pregnant rhesus monkeys were exposed to filtered air (FA) or SHS (1 mg/m{sup 3} total suspended particulates) for 6 h/day, 5 days/week starting at 50 days gestational age. The mother/infant pairs continued the SHS exposures postnatally. At 3 months of age each infant: 1) had in vivo lung function measurements in response to inhaled methacholine, or 2) the right accessory lobe filled with agarose, precision-cut to 600 {mu}m slices, and bathed in increasing concentrations of methacholine. The lumenal area of the central airway was determined using videomicrometry followed by fixation and histology with morphometry. In vivo tests showed that perinatal SHS increases baseline respiratory rate and decreases responsiveness to methacholine. Perinatal SHS did not alter intrinsic airway responsiveness in the bronchi. However in respiratory bronchioles, SHS exposure increased airway responsiveness at lower methacholine concentrations but decreased it at higher concentrations. Perinatal SHS did not change eosinophil profiles, epithelial volume, smooth muscle volume, or mucin volume. However it did increase the number of alveolar attachments in bronchi and respiratory bronchioles. In general, as mucin increased, airway responsiveness decreased. We conclude that perinatal SHS exposure alters in vivo and intrinsic airway responsiveness, and alveolar attachments.

  16. In vivo kinetic analysis of the penicillin biosynthesis pathway using PAA stimulus response experiments.

    PubMed

    Deshmukh, Amit T; Verheijen, Peter J T; Maleki Seifar, Reza; Heijnen, Joseph J; van Gulik, Walter M

    2015-11-01

    In this study we combined experimentation with mathematical modeling to unravel the in vivo kinetic properties of the enzymes and transporters of the penicillin biosynthesis pathway in a high yielding Penicillium chrysogenum strain. The experiment consisted of a step response experiment with the side chain precursor phenyl acetic acid (PAA) in a glucose-limited chemostat. The metabolite data showed that in the absence of PAA all penicillin pathway enzymes were expressed, leading to the production of a significant amount of 6-aminopenicillanic acid (6APA) as end product. After the stepwise perturbation with PAA, the pathway produced PenG within seconds. From the extra- and intracellular metabolite measurements, hypotheses for the secretion mechanisms of penicillin pathway metabolites were derived. A dynamic model of the penicillin biosynthesis pathway was then constructed that included the formation and transport over the cytoplasmic membrane of pathway intermediates, PAA and the product penicillin-G (PenG). The model parameters and changes in the enzyme levels of the penicillin biosynthesis pathway under in vivo conditions were simultaneously estimated using experimental data obtained at three different timescales (seconds, minutes, hours). The model was applied to determine changes in the penicillin pathway enzymes in time, calculate fluxes and analyze the flux control of the pathway. This led to a reassessment of the in vivo behavior of the pathway enzymes and in particular Acyl-CoA:Isopenicillin N Acyltransferase (AT). PMID:26476338

  17. Model-based analysis of clinical fluorescence spectroscopy for in vivo detection of cervical intraepithelial dysplasia

    NASA Astrophysics Data System (ADS)

    Chang, Sung K.; Marín, Nena; Follen, Michelle; Richards-Kortum, Rebecca R.

    2006-03-01

    We present a mathematical model to calculate the relative concentration of light scatterers, light absorbers, and fluorophores in the epithelium and stroma. This mathematical description is iteratively fit to the fluorescence spectra measured in vivo, yielding relative concentrations of each molecule. The mathematical model is applied to a total of 493 fluorescence measurements of normal and dysplastic cervical tissue acquired in vivo from 292 patients. The estimated parameters are compared with histopathologic diagnosis to evaluate their diagnostic potential. The mathematical model is validated using fluorescence spectra simulated with known sets of optical parameters. Subsequent application of the mathematical model to in vivo fluorescence measurements from cervical tissue yields fits that accurately describe measured data. The optical parameters estimated from 493 fluorescence measurements show an increase in epithelial flavin adenine dinucleotide (FAD) fluorescence, a decrease in epithelial keratin fluorescence, an increase in epithelial light scattering, a decrease in stromal collagen fluorescence, and an increase in stromal hemoglobin light absorption in dysplastic tissue compared to normal tissue. These changes likely reflect an increase in the metabolic activity and loss of differentiation of epithelial dysplastic cells, and stromal angiogenesis associated with dysplasia. The model presented here provides a tool to analyze clinical fluorescence spectra yielding quantitative information about molecular changes related to dysplastic transformation.

  18. Live-cell high resolution magic angle spinning magnetic resonance spectroscopy for in vivo analysis of Pseudomonas aeruginosa metabolomics

    PubMed Central

    RIGHI, VALERIA; CONSTANTINOU, CATERINA; KESARWANI, MEENU; RAHME, LAURENCE G.; TZIKA, ARIA A.

    2013-01-01

    Pseudomonas aeruginosa (PA) is a pathogenic gram-negative bacterium that is widespread in nature, inhabiting soil, water, plants and animals. PA is a prevalent cause of deleterious human infections, particularly in patients whose host defense mechanisms have been compromised. Metabolomics is an important tool used to study host-pathogen interactions and to identify novel therapeutic targets and corresponding compounds. The aim of the present study was to report the metabolic profile of live PA bacteria using in vivo high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance spectroscopy (NMR), in combination with 1- and 2-dimensional HRMAS NMR. This methodology provides a new and powerful technique to rapidly interrogate the metabolome of intact bacterial cells and has several advantages over traditional techniques that identify metabolome components from disrupted cells. Furthermore, application of multidimensional HRMAS NMR, in combination with the novel technique total through-Bond correlation Spectroscopy (TOBSY), is a promising approach that may be used to obtain in vivo metabolomics information from intact live bacterial cells and can mediate such analyses in a short period of time. Moreover, HRMAS 1H NMR enables the investigation of the associations between metabolites and cell processes. In the present study, we detected and quantified several informative metabolic molecules in live PA cells, including N-acetyl, betaine, citrulline, alanine and glycine, which are important in peptidoglycan synthesis. The results provided a complete metabolic profile of PA for future studies of PA clinical isolates and mutants. In addition, this in vivo NMR biomedical approach might have clinical utility and should prove useful in gene function validation, the study of pathogenetic mechanisms, the classification of microbial strains into functional/clinical groups, the testing of anti-bacterial agents and the determination of metabolic profiles of bacterial mutants. PMID:24649014

  19. Trial-Based Functional Analysis and Functional Communication Training in an Early Childhood Setting

    ERIC Educational Resources Information Center

    Lambert, Joseph M.; Bloom, Sarah E.; Irvin, Jennifer

    2012-01-01

    Problem behavior is common in early childhood special education classrooms. Functional communication training (FCT; Carr & Durand, 1985) may reduce problem behavior but requires identification of its function. The trial-based functional analysis (FA) is a method that can be used to identify problem behavior function in schools. We conducted…

  20. Analysis of the interaction of phytoestrogens and synthetic chemicals: An in vitro/in vivo comparison

    SciTech Connect

    Charles, Grantley D. . E-mail: charles_grantley@allergan.com; Gennings, Chris; Tornesi, Belen; Kan, H. Lynn; Zacharewski, Timothy R.; Bhaskar Gollapudi, B.; Carney, Edward W.

    2007-02-01

    In the evaluation of chemical mixture toxicity, it is desirable to develop an evaluation paradigm which incorporates some critical attributes of real world exposures, particularly low dose levels, larger numbers of chemicals, and chemicals from synthetic and natural sources. This study evaluated the impact of low level exposure to a mixture of six synthetic chemicals (SC) under conditions of co-exposure to various levels of plant-derived phytoestrogen (PE) compounds. Estrogenic activity was evaluated using an in vitro human estrogen receptor (ER) transcriptional activation assay and an in vivo immature rat uterotrophic assay. Initially, dose-response curves were characterized for each of the six SCs (methoxyclor, o,p-DDT, octylphenol, bisphenol A, {beta}-hexachlorocyclohexane, 2,3-bis(4-hydroxyphenyl)-propionitrile) in each of the assays. The six SCs were then combined at equipotent ratios and tested at 5-6 dose levels spanning from very low, sub-threshold levels, to a dose in which every chemical in the mixture was at its individual estrogenic response threshold. The SC mixtures also were tested in the absence or presence of 5-6 different levels of PEs, for a total of 36 (in vitro) or 25 (in vivo) treatment groups. Both in vitro and in vivo, low concentrations of the SC mixture failed to increase estrogenic responses relative to those induced by PEs alone. However, significant increases in response occurred when each chemical in the SC mixture was near or above its individual response threshold. In vitro, interactions between high-doses of SCs and PEs were greater than additive, whereas mixtures of SCs in the absence of PEs interacted in a less than additive fashion. In vivo, the SC and PE mixture responses were consistent with additivity. These data illustrate a novel approach for incorporating key attributes of real world exposures in chemical mixture toxicity assessments, and suggest that chemical mixture toxicity is likely to be of concern only when the mixture components are near or above their individual response thresholds. However, these data suggest that extrapolation from in vitro assays to in vivo mixture effects should be approached with caution.

  1. Fluorescently Tagged Potato virus Y: A Versatile Tool for Functional Analysis of Plant-Virus Interactions.

    PubMed

    Matevz, Rupar; Florence, Faurez; Michel, Tribodet; Ion, Gutiérrez-Aguirre; Agnčs, Delaunay; Laurent, Glais; Maja, Kriznik; David, Dobnik; Kristina, Gruden; Emmanuel, Jacquot; Maja, Ravnikar

    2015-07-01

    Potato virus Y (PVY) is an economically important plant virus that infects Solanaceous crops such as tobacco and potato. To date, studies into the localization and movement of PVY in plants have been limited to detection of viral RNA or proteins ex vivo. Here, a PVY N605 isolate was tagged with green fluorescent protein (GFP), characterized and used for in vivo tracking. In Nicotiana tabacum cv. Xanthi, PVY N605-GFP was biologically comparable to nontagged PVY N605, stable through three plant-to-plant passages and persisted for four months in infected plants. GFP was detected before symptoms and fluorescence intensity correlated with PVY RNA concentrations. PVY N605-GFP provided in vivo tracking of long-distance movement, allowing estimation of the cell-to-cell movement rate of PVY in N. tabacum cv. Xanthi (7.1 ± 1.5 cells per hour). PVY N605-GFP was adequately stable in Solanum tuberosum cvs. Désirée and NahG-Désirée and able to infect S. tuberosum cvs. Bintje and Bea, Nicotiana benthamiana, and wild potato relatives. PVY N605-GFP is therefore a powerful tool for future studies of PVY-host interactions, such as functional analysis of viral and plant genes involved in viral movement. PMID:25761209

  2. Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism.

    PubMed Central

    Umezu, K; Sugawara, N; Chen, C; Haber, J E; Kolodner, R D

    1998-01-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages. PMID:9539419

  3. Cadmium concentration in the kidney cortex of occupationally exposed workers measured in vivo using X-ray fluorescence analysis

    SciTech Connect

    Christoffersson, J.O.; Welinder, H.; Spang, G.M.; Mattsson, S.; Skerfving, S.

    1987-04-01

    A method for in vivo X-ray fluorescence analysis of the cadmium concentration in the kidney cortex has been improved and tested in 20 selected male occupationally cadmium-exposed workers (duration of exposure 7-39 years). The concentration of cadmium in kidney cortex ranged from 47 to 317 (median 141) micrograms/g. The concentration of cadmium in blood was 32-160 (median 64) nmole/liter, cadmium in urine was 2.5-13 (median 5.4) nmole/mmole creatinine, and beta 2-microglobulin in urine was 3.3-68 (median 14) micrograms/mmole creatinine. In individuals, the relationship between duration of exposure, time-integrated exposure, and cadmium level in urine on the one hand and cadmium level in the kidney on the other varied considerably. Direct in vivo analysis of the concentration of cadmium in the kidney cortex is therefore valuable as a complement to the other tests when monitoring cadmium exposure. Our method is sensitive, practically free from risk, and can b performed by routine at low costs.

  4. Contractile behavior of the forelimb digital flexors during steady-state locomotion in horses (Equus caballus): an initial test of muscle architectural hypotheses about in vivo function.

    PubMed

    Butcher, M T; Hermanson, J W; Ducharme, N G; Mitchell, L M; Soderholm, L V; Bertram, J E A

    2009-01-01

    The forelimb digital flexors of the horse display remarkable diversity in muscle architecture despite each muscle-tendon unit having a similar mechanical advantage across the fetlock joint. We focus on two distinct muscles of the digital flexor system: short compartment deep digital flexor (DDF(sc)) and the superficial digital flexor (SDF). The objectives were to investigate force-length behavior and work performance of these two muscles in vivo during locomotion, and to determine how muscle architecture contributes to in vivo function in this system. We directly recorded muscle force (via tendon strain gauges) and muscle fascicle length (via sonomicrometry crystals) as horses walked (1.7 m s(-1)), trotted (4.1 m s(-1)) and cantered (7.0 m s(-1)) on a motorized treadmill. Over the range of gaits and speeds, DDF(sc) fascicles shortened while producing relatively low force, generating modest positive net work. In contrast, SDF fascicles initially shortened, then lengthened while producing high force, resulting in substantial negative net work. These findings suggest the long fibered, unipennate DDF(sc) supplements mechanical work during running, whereas the short fibered, multipennate SDF is specialized for economical high force and enhanced elastic energy storage. Apparent in vivo functions match well with the distinct architectural features of each muscle. PMID:18835360

  5. Ex vivo promoter analysis of antiviral heat shock cognate 70B gene in Anopheles gambiae

    PubMed Central

    Kang, Seokyoung; Sim, Cheolho; Byrd, Brian D; Collins, Frank H; Hong, Young S

    2008-01-01

    Background The Anopheles gambiae heat shock cognate gene (hsc70B) encodes a constitutively expressed protein in the hsp70 family and it functions as a molecular chaperone for protein folding. However, the expression of hsc70B can be further induced by certain stimuli such as heat shock and infection. We previously demonstrated that the An. gambiae hsc70B is induced during o'nyong-nyong virus (ONNV) infection and subsequently suppresses ONNV replication in the mosquito. To further characterize the inducibility of hsc70B by ONNV infection in An. gambiae, we cloned a 2.6-kb region immediately 5' upstream of the starting codon of hsc70B into a luciferase reporter vector (pGL3-Basic), and studied its promoter activity in transfected Vero cells during infection with o'nyong-nyong, West Nile and La Crosse viruses. Results Serial deletion analysis of the hsc70B upstream sequence revealed that the putative promoter is likely located in a region 1615–2150 bp upstream of the hsc70B starting codon. Sequence analysis of this region revealed transcriptional regulatory elements for heat shock element-binding protein (HSE-bind), nuclear factor ?B (NF-?B), dorsal (Dl) and fushi-tarazu (Ftz). Arbovirus infection, regardless of virus type, significantly increased the hsc70B promoter activity in transfected Vero cells. Conclusion Our results further validate the transcriptional activation of hsc70B during arbovirus infection and support the role of specific putative regulatory elements. Induction by three taxonomically distinct arboviruses suggests that the HSC70B protein may be expressed to cope with cellular stress imposed during infection. PMID:18986525

  6. Analysis of exercise-induced Na+-K+ exchange in rat skeletal muscle in vivo.

    PubMed

    Murphy, K T; Nielsen, O B; Clausen, T

    2008-12-01

    We aimed to quantify the Na(+)-K(+) exchange occurring during exercise in rat skeletal muscle in vivo. Intracellular Na(+) and K(+) content, Na(+) permeability ((22)Na(+) influx), Na(+)-K(+) pump activity (ouabain-sensitive (86)Rb(+) uptake) and Na(+)-K(+) pump alpha(2) subunit content ([(3)H]ouabain binding) were measured. Six-week-old rats rested (control animals) or performed intermittent running for 10-60 min and were then killed or were killed at 15 or 90 min following 60 min exercise. In the soleus muscle, intracellular Na(+) was 80% higher than in control rats after 60 min exercise, was still elevated (38%) after 15 min rest and returned to control levels after 90 min rest. Intracellular K(+) showed corresponding decreases after 15-60 min exercise, returning to control levels 90 min postexercise. Exercise induced little change in Na(+) and K(+) in the extensor digitorum longus muscle (EDL). In soleus, the exercise-induced rise in Na(+) and reduction in K(+) were augmented by pretreatment with ouabain or by reducing the content of muscular Na(+)-K(+) pumps by prior K(+) depletion of the animals. Fifteen minutes after 60 min exercise, ouabain-sensitive (86)Rb(+) uptake in the soleus was increased by 30% but was unchanged in EDL, and there was no effect of exercise on [(3)H]ouabain binding measured in vitro or in vivo in either muscle. In conclusion, in the soleus, in vivo exercise induces a rise in intracellular Na(+), which reflects the excitation-induced increase in Na(+) influx and leads to augmented Na(+)-K(+) pump activity without apparent change in Na(+)-K(+) pump capacity. PMID:18586859

  7. Alterations to Functional Analysis Methodology to Clarify the Functions of Low Rate, High Intensity Problem Behavior

    PubMed Central

    Davis, Barbara J; Kahng, SungWoo; Schmidt, Jonathan; Bowman, Lynn G; Boelter, Eric W

    2012-01-01

    Current research provides few suggestions for modifications to functional analysis procedures to accommodate low rate, high intensity problem behavior. This study examined the results of the extended duration functional analysis procedures of Kahng, Abt, and Schonbachler (2001) with six children admitted to an inpatient hospital for the treatment of severe problem behavior. Results of initial functional analyses (Iwata, Dorsey, Slifer, Bauman, & Richman, 1982/1994) were inconclusive for all children because of low levels of responding. The altered functional analyses, which changed multiple variables including the duration of the functional analysis (i.e., 6 or 7 hrs), yielded clear behavioral functions for all six participants. These results add additional support for the utility of an altered analysis of low rate, high intensity problem behavior when standard functional analyses do not yield differentiated results. PMID:23326628

  8. The Saccharomyces cerevisiae MGT1 DNA repair methyltransferase gene: its promoter and entire coding sequence, regulation and in vivo biological functions.

    PubMed Central

    Xiao, W; Samson, L

    1992-01-01

    We previously cloned a yeast DNA fragment that, when fused with the bacterial lacZ promoter, produced O6-methylguanine DNA repair methyltransferase (MGT1) activity and alkylation resistance in Escherichia coli (Xiao et al., EMBO J. 10,2179). Here we describe the isolation of the entire MGT1 gene and its promoter by sequence directed chromosome integration and walking. The MGT1 promoter was fused to a lacZ reporter gene to study how MGT1 expression is controlled. MGT1 is not induced by alkylating agents, nor is it induced by other DNA damaging agents such as UV light. However, deletion analysis defined an upstream repression sequence, whose removal dramatically increased basal level gene expression. The polypeptide deduced from the complete MGT1 sequence contained 18 more N-terminal amino acids than that previously determined; the role of these 18 amino acids, which harbored a potential nuclear localization signal, was explored. The MGT1 gene was also cloned under the GAL1 promoter, so that MTase levels could be manipulated, and we examined MGT1 function in a MTase deficient yeast strain (mgt1). The extent of resistance to both alkylation-induced mutation and cell killing directly correlated with MTase levels. Finally we show that mgt1 S.cerevisiae has a higher rate of spontaneous mutation than wild type cells, indicating that there is an endogenous source of DNA alkylation damage in these eukaryotic cells and that one of the in vivo roles of MGT1 is to limit spontaneous mutations. PMID:1641326

  9. Analysis of green fluorescent protein bioluminescence in vivo and in vitro using a glow discharge

    NASA Astrophysics Data System (ADS)

    Hernández, L.; Mandujano, L. A.; Cuevas, J.; Reyes, P. G.; Osorio-González, D.

    2015-03-01

    The discovery of fluorescent proteins has been a revolution in cell biology and related sciences because of their many applications, mainly emphasizing their use as cellular markers. The green fluorescent protein (GFP) is one of the most used as it requires no cofactors to generate fluorescence and retains this property into any organism when it is expressed by recombinant DNA techniques, which is a great advantage. In this work, we analyze the emission spectra of recombinant green fluorescent protein in vivo and in vitro exposed to a glow discharge plasma of nitrogen in order to relate electron temperature to fluorescence intensity.

  10. Network Analysis of Intrinsic Functional Brain Connectivity in Alzheimer's Disease

    E-print Network

    Rubin, Daniel L.

    Network Analysis of Intrinsic Functional Brain Connectivity in Alzheimer's Disease Kaustubh Supekar organization is disrupted in Alzheimer's disease (AD). Task-free fMRI data from 21 AD subjects and 18 age Analysis of Intrinsic Functional Brain Connectivity in Alzheimer's Disease. PLoS Comput Biol 4(6): e1000100

  11. An exploration of function analysis and function allocation in the commercial flight domain

    NASA Technical Reports Server (NTRS)

    Mcguire, James C.; Zich, John A.; Goins, Richard T.; Erickson, Jeffery B.; Dwyer, John P.; Cody, William J.; Rouse, William B.

    1991-01-01

    The applicability is explored of functional analysis methods to support cockpit design. Specifically, alternative techniques are studied for ensuring an effective division of responsibility between the flight crew and automation. A functional decomposition is performed of the commercial flight domain to provide the information necessary to support allocation decisions and demonstrate methodology for allocating functions to flight crew or to automation. The function analysis employed 'bottom up' and 'top down' analyses and demonstrated the comparability of identified functions, using the 'lift off' segment of the 'take off' phase as a test case. The normal flight mission and selected contingencies were addressed. Two alternative methods for using the functional description in the allocation of functions between man and machine were investigated. The two methods were compared in order to ascertain their relative strengths and weaknesses. Finally, conclusions were drawn regarding the practical utility of function analysis methods.

  12. Wannier function analysis of silicon carbon alloys

    NASA Astrophysics Data System (ADS)

    Fitzhenry, P.; Bilek, M. M. M.; Marks, N. A.; Cooper, N. C.; McKenzie, D. R.

    2003-01-01

    Maximally localized Wannier functions are the basis of a new technique for resolving ambiguous bonding issues for amorphous materials. Geometrical methods using the Wannier function representation provide an insightful chemical picture of local bonding and hybridization in disordered structures. Central to these methods is the notion of treating the Wannier function centres as a virtual atomic species with a well-defined degree of localization. Using Wannier function methods, we classify and quantify the types of bonding present in a sample of the ternary alloy hydrogenated amorphous silicon carbide, C22Si22H20. In addition to the bonding previously observed for this material, we see three-centre bonding and flipping bonds. We identify a cluster defect in our sample associated with these flipping bonds, and observe a temperature dependence of the bond flipping. This effect may be observable using temperature-dependent Raman spectroscopy.

  13. In vivo imaging and quantitative analysis of zebrafish embryos by digital holographic microscopy

    PubMed Central

    Gao, Jian; Lyon, Joseph A.; Szeto, Daniel P.; Chen, Jun

    2012-01-01

    Digital holographic microscopy (DHM) has been applied extensively to in vitro studies of different living cells. In this paper, we present a novel application of an off-axis DHM system to in vivo study of the development of zebrafish embryos. Even with low magnification microscope objectives, the morphological structures and individual cell types inside developing zebrafish embryos can be clearly observed from reconstructed amplitude images. We further study the dynamic process of blood flow in zebrafish embryos. A calibration routine and post-processing procedures are developed to quantify physiological parameters at different developmental stages. We measure quantitatively the blood flow as well as the heart rate to study the effects of elevated D-glucose (abnormal condition) on circulatory and cardiovascular systems of zebrafish embryos. To enhance our ability to use DHM as a quantitative tool for potential high throughput screening application, the calibration and post-processing algorithms are incorporated into an automated processing software. Our results show that DHM is an excellent non-invasive imaging technique for visualizing the cellular dynamics of organogenesis of zebrafish embryos in vivo. PMID:23082301

  14. Analysis of in vivo penetration of textile dyes causing allergic reactions

    NASA Astrophysics Data System (ADS)

    Lademann, J.; Patzelt, A.; Worm, M.; Richter, H.; Sterry, W.; Meinke, M.

    2009-10-01

    Contact allergies to textile dyes are common and can cause severe eczema. In the present study, we investigated the penetration of a fluorescent textile dye, dissolved from a black pullover, into the skin of one volunteer during perspiration and nonperspiration. Previously, wearing this pullover had induced a severe contact dermatitis in an 82-year old woman, who was not aware of her sensitization to textile dyes. The investigations were carried out by in vivo laser scanning microscopy. It could be demonstrated that the dye was eluted from the textile material by sweat. Afterwards, the dye penetrated into the stratum corneum and into the hair follicles. Inside the hair follicles, the fluorescent signal was still detectable after 24 h, whereas it was not verifiable anymore in the stratum corneum, Laser scanning microscopy represents an efficient tool for in vivo investigation of the penetration and storage of topically applied substances and allergens into the human skin and reveals useful hints for the development and optimization of protection strategies.

  15. Hybrid System for Ex Vivo Hemorheological and Hemodynamic Analysis: A Feasibility Study

    PubMed Central

    Yeom, Eunseop; Jun Kang, Yang; Joon Lee, Sang

    2015-01-01

    Precise measurement of biophysical properties is important to understand the relation between these properties and the outbreak of cardiovascular diseases (CVDs). However, a systematic measurement for these biophysical parameters under in vivo conditions is nearly impossible because of complex vessel shape and limited practicality. In vitro measurements can provide more biophysical information, but in vitro exposure changes hemorheological properties. In this study, a hybrid system composed of an ultrasound system and microfluidic device is proposed for monitoring hemorheological and hemodynamic properties under more reasonable experimental conditions. Biophysical properties including RBC aggregation, viscosity, velocity, and pressure of blood flows are simultaneously measured under various conditions to demonstrate the feasibility and performance of this measurement system. The proposed technique is applied to a rat extracorporeal loop which connects the aorta and jugular vein directly. As a result, the proposed system is found to measure biophysical parameters reasonably without blood collection from the rat and provided more detailed information. This hybrid system, combining ultrasound imaging and microfluidic techniques to ex vivo animal models, would be useful for monitoring the variations of biophysical properties induced by chemical agents. It can be used to understand the relation between biophysical parameters and CVDs. PMID:26090816

  16. RIVET-a tool for in vivo analysis of symbiotically relevant gene expression in Sinorhizobium meliloti.

    PubMed

    Gao, Mengsheng; Teplitski, Max

    2008-02-01

    Despite significant advances in the development of sensitive tools for studying genetics and signal exchange in legume-rhizobium symbioses, many uncertainties remain about the in vivo role of bacterial and plant signals in symbiotic gene regulation. In this study, we adapted TnpR recombinase-based in vivo expression technology (RIVET) to document gene regulation in Sinorhizobium meliloti. The substrate for TnpR, the res1-tet-res1 cassette, is stably inherited when cloned into a neutral site of the S. meliloti genome. Bicistronic promoterless tnpR-beta-glucuronidase (GUS) reporters were constructed to track expression ("resolution") of symbiotically relevant S. meliloti genes during different stages of the interaction. In proof of principle experiments, the resolution of the nodC::tnpR reporter was detected within 4 h of exposure to micromolar levels of the nod operon inducer luteolin and after overnight incubation in the rhizosphere. RIVET demonstrated that cell division gene ftsZ2 was not strongly expressed in the rhizosphere but was activated inside the nodules and on agar surfaces. Rhizosphere expression of the N-acyl homoserine lactone (AHL) synthase sinI::tnpR-GUS reporter was modest in prequorate microcolonies, and then increased with time. AHL synthase sinI and an AHL-regulated gene, expG, were activated inside the nodules. PMID:18184060

  17. Genome Analysis Maintenance of duplicate genes and their functional

    E-print Network

    Zhang, Jianzhi

    Genome Analysis Maintenance of duplicate genes and their functional redundancy by reduced- gence between duplicate genes, many old duplicates still maintain a high degree of functional similarity- tion of their ancestral functions. Consistent with this hypothesis, gene expression data from both

  18. Application of Lie group analysis to functional differential equations

    E-print Network

    Martin Oberlack; Marta Waclawczyk

    2006-10-27

    In the present paper the classical point symmetry analysis is extended from partial differential to functional differential equations with functional derivatives. In order to perform the group analysis and deal with the functional derivatives we extend the quantities such as infinitesimal transformations, prolongations and invariant solutions. For the sake of example the procedure is applied to the continuum limit of the heat equation. The method can further lead to significant applications in statistical physics and fluid dynamics.

  19. In vitro and in vivo analysis of antimicrobial agents alone and in combination against multi-drug resistant Acinetobacter baumannii

    PubMed Central

    He, Songzhe; He, Hui; Chen, Yi; Chen, Yueming; Wang, Wei; Yu, Daojun

    2015-01-01

    Objective: To investigate the in vitro and in vivo antibacterial activities of tigecycline and other 13 common antimicrobial agents, alone or in combination, against multi-drug resistant Acinetobacter baumannii. Methods: An in vitro susceptibility test of 101 A. baumannii was used to detect minimal inhibitory concentrations (MICs). A mouse lung infection model of multi-drug resistant A. baumannii, established by the ultrasonic atomization method, was used to define in vivo antimicrobial activities. Results: Multi-drug resistant A. baumannii showed high sensitivity to tigecycline (98% inhibition), polymyxin B (78.2% inhibition), and minocycline (74.2% inhibition). However, the use of these antimicrobial agents in combination with other antimicrobial agents produced synergistic or additive effects. In vivo data showed that white blood cell (WBC) counts in drug combination groups C (minocycline + amikacin) and D (minocycline + rifampicin) were significantly higher than in groups A (tigecycline) and B (polymyxin B) (P < 0.05), after administration of the drugs 24 h post-infection. Lung tissue inflammation gradually increased in the model group during the first 24 h after ultrasonic atomization infection; vasodilation, congestion with hemorrhage were observed 48 h post infection. After 3 days of anti-infective therapy in groups A, B, C, and D, lung tissue inflammation in each group gradually recovered with clear structures. The mortality rates in drug combination groups(groups C and D) were much lower than in groups A and B. Conclusion: The combination of minocycline with either rifampicin or amikacin is more effective against multi-drug resistant A. baumannii than single-agent tigecycline or polymyxin B. In addition, the mouse lung infection by ultrasonic atomization is a suitable model for drug screening and analysis of infection mechanism. PMID:26074898

  20. Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins.

    PubMed

    Junge, Friederike; Haberstock, Stefan; Roos, Christian; Stefer, Susanne; Proverbio, Davide; Dötsch, Volker; Bernhard, Frank

    2011-04-30

    Cell-free expression has emerged as a powerful technique to overcome major restrictions of classical in vivo membrane protein production, with sample yields of mgms of protein per ml reaction volume possible in less than a day. The open nature and high versatility of cell-free expression allows a variety of completely new ways to rationally design and optimise expression environments as well as to modulate folding kinetics for membrane proteins independent of their origin, size, topology and function. This article summarises the array of currently available options to modify and develop cell-free expression protocols adapted to the specific requirements of individual membrane proteins. We give further an overview of the recent advances of cell-free production of membrane proteins for structural and functional analysis. PMID:20637904

  1. Mutational and Functional Analysis Reveals ADAMTS18 Metalloproteinase as a Novel Oncogene in Melanoma

    PubMed Central

    Wei, Xiaomu; Prickett, Todd D.; Viloria, Cristina G.; Molinolo, Alfredo; Lin, Jimmy C.; Cardenas-Navia, Isabel; Cruz, Pedro; Rosenberg, Steven A.; Davies, Michael A.; Gershenwald, Jeffrey E.; López-Otín, Carlos; Samuels, Yardena

    2010-01-01

    The disintegrin-metalloproteinases with thrombospondin domains (ADAMTS) genes have been suggested to function as tumor suppressors as several have been found to be epigenetically silenced in various cancers. We performed a mutational analysis of the ADAMTS gene family in human melanoma and identified a large fraction of melanomas to harbor somatic mutations. To evaluate the functional consequences of the most commonly mutated gene, ADAMTS18, six of its mutations were biologically examined. ADAMTS18 mutations had little effect on melanoma cell growth under standard conditions, but reduced cell dependence on growth factors. ADAMTS18 mutations also reduced adhesion to laminin and increased migration in vitro and metastasis in vivo. Melanoma cells expressing mutant ADAMTS18 had reduced cell migration after shRNA-mediated knockdown of ADAMTS18, suggesting that ADAMTS18 mutations are growth-, migration- and metastasis- promoting in melanoma. PMID:21047771

  2. False Positive Functional Analysis Results as a Contributor of Treatment Failure during Functional Communication Training

    ERIC Educational Resources Information Center

    Mann, Amanda J.; Mueller, Michael M.

    2009-01-01

    Research has shown that functional analysis results are beneficial for treatment selection because they identify reinforcers for severe behavior that can then be used to reinforce replacement behaviors either differentially or noncontingently. Theoretically then, if a reinforcer is identified in a functional analysis erroneously, a well researched…

  3. Analysis of the structure of Tetrahymena nuclear RNAs in vivo: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA.

    PubMed

    Zaug, A J; Cech, T R

    1995-06-01

    Dimethyl sulfate modification of RNA in living Tetrahymena thermophila allowed assessment of RNA secondary structure and protein association. The self-splicing rRNA intron had the same methylation pattern in vivo as in vitro, indicating that the structures are equivalent and suggesting that this RNA is not stably associated with protein in the nucleolus. Methylation was consistent with the current secondary structure model. Much of telomerase RNA was protected from methylation in vivo, but the A's and C's in the template region were very reactive. Thus, most telomerase is not base paired to telomeres in vivo. Protein-free telomerase RNA adopts a structure different from that in vivo, especially in the template and pseudoknot regions. The U2 snRNA showed methylation protection at the Sm protein-binding sequence and the mRNA branch site recognition sequence. For both telomerase RNA and U2 snRNA, the in vivo methylation pattern corresponded much better to the structure determined by comparative sequence analysis than did the in vitro methylation pattern. Thus, as expected, comparative analysis gives the structure of the RNA in vivo. PMID:7493315

  4. Acceleration of reverse analysis method using hyperbolic activation function

    NASA Astrophysics Data System (ADS)

    Pwasong, Augustine; Sathasivam, Saratha

    2015-10-01

    Hyperbolic activation function is examined for its ability to accelerate the performance of doing data mining by using a technique named as Reverse Analysis method. In this paper, we describe how Hopfield network perform better with hyperbolic activation function and able to induce logical rules from large database by using reverse analysis method: given the values of the connections of a network, we can hope to know what logical rules are entrenched in the database. We limit our analysis to Horn clauses.

  5. Preserved endothelium-dependent dilatation of the coronary microvasculature at the early phase of diabetes mellitus despite the increased oxidative stress and depressed cardiac mechanical function ex vivo

    PubMed Central

    2013-01-01

    Background There has been accumulating evidence associating diabetes mellitus and cardiovascular dysfunctions. However, most of the studies are focused on the late stages of diabetes and on the function of large arteries. This study aimed at characterizing the effects of the early phase of diabetes mellitus on the cardiac and vascular function with focus on the intact coronary microvasculature and the oxidative stress involved. Materials and methods Zucker diabetic fatty rats and their lean littermates fed with standard diet A04 (Safe) were studied at the 11th week of age. Biochemical parameters such as glucose, insulin and triglycerides levels as well as their oxidative stress status were measured. Their hearts were perfused ex vivo according to Langendorff and their cardiac activity and coronary microvascular reactivity were evaluated. Results Zucker fatty rats already exhibited a diabetic state at this age as demonstrated by the elevated levels of plasma glucose, insulin, glycated hemoglobin and triglycerides. The ex vivo perfusion of their hearts revealed a decreased cardiac mechanical function and coronary flow. This was accompanied by an increase in the overall oxidative stress of the organs. However, estimation of the active form of endothelial nitric oxide synthase and coronary reactivity indicated a preserved function of the coronary microvessels at this phase of the disease. Diabetes affected also the cardiac membrane phospholipid fatty acid composition by increasing the arachidonic acid and n-3 polyunsaturated fatty acids levels. Conclusions The presence of diabetes, even at its beginning, significantly increased the overall oxidative stress of the organs resulting to decreased cardiac mechanical activity ex vivo. However, adaptations were adopted at this early phase of the disease regarding the preserved coronary microvascular reactivity and the associated cardiac phospholipid composition in order to provide a certain protection to the heart. PMID:23530768

  6. Single-walled carbon nanotubes noncovalently functionalized with lipid modified polyethylenimine for siRNA delivery in vitro and in vivo.

    PubMed

    Siu, King S; Zheng, Xiufen; Liu, Yanling; Zhang, Yujuan; Zhang, Xusheng; Chen, Di; Yuan, Ken; Gillies, Elizabeth R; Koropatnick, James; Min, Wei-Ping

    2014-10-15

    siRNA can downregulate the expression of specific genes. However, delivery to specific cells and tissues in vivo presents significant challenges. Modified carbon nanotubes (CNTs) have been shown to protect siRNA and facilitate its entry into cells. However, simple and efficient methods to functionalize CNTs are needed. Here, noncovalent functionalization of CNTs is performed and shown to effectively deliver siRNA to target cells. Specifically, single-walled CNTs were functionalized by noncovalent association with a lipopolymer. The lipopolymer (DSPE-PEG) was composed of a phospholipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and poly(ethylene glycol) (PEG). Three different ratios of polyethylenimine (PEI) to DSPE-PEG were synthesized and characterized and the products were used to disperse CNTs. The resulting materials were used for siRNA delivery in vitro and in vivo. The structural, biophysical, and biological properties of DGI/C and their complexes formed with siRNA were investigated. Cytotoxicity of the materials was low, and effective gene silencing in B16-F10 cells was demonstrated in vitro. In addition, significant uptake of siRNA as well as gene silencing in the liver was found following intravenous injection. This approach provides a new strategy for siRNA delivery and could provide insight for the development of noncovalently functionalized CNTs for siRNA therapy. PMID:25216445

  7. Barbara MacCluer: Elementary Functional Analysis. Books on Functional Analysis are not exactly ubiquitous, but every publishing

    E-print Network

    Werner, Dirk

    Barbara MacCluer: Elementary Functional Analysis. Books on Functional Analysis are not exactly ubiquitous, but every publishing house has several text books in this area on its backlist. Barbara MacCluer's book is an excellent new entry. The book consists of six chapters. Chapter 1 introduces

  8. In Vitro Mutational Analysis of the ?2 Adrenergic Receptor, an In Vivo Surrogate Odorant Receptor

    PubMed Central

    Pfister, Patrick; Tomoiaga, Delia; Rogers, Matthew E.; Feinstein, Paul

    2015-01-01

    Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse ?2-adrenergic-receptor (m?2AR), robustly traffics to the plasma membrane. We set out to characterize m?2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of m?2AR using a Green Fluorescent Protein-tagged m?2AR (m?2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known m?2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of m?2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of m?2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three m?2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower m?2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs. PMID:26513247

  9. In Vitro Mutational Analysis of the ?2 Adrenergic Receptor, an In Vivo Surrogate Odorant Receptor.

    PubMed

    Jamet, Sophie; Bubnell, Jaclyn; Pfister, Patrick; Tomoiaga, Delia; Rogers, Matthew E; Feinstein, Paul

    2015-01-01

    Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse ?2-adrenergic-receptor (m?2AR), robustly traffics to the plasma membrane. We set out to characterize m?2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of m?2AR using a Green Fluorescent Protein-tagged m?2AR (m?2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known m?2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of m?2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of m?2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three m?2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower m?2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs. PMID:26513247

  10. Systemic Analysis of Regulated Functional Networks.

    PubMed

    Hernández Sánchez, Luis Francisco; Aasebř, Elise; Selheim, Frode; Berven, Frode S; Rćder, Helge; Barsnes, Harald; Vaudel, Marc

    2016-01-01

    In biological and medical sciences, high throughput analytical methods are now commonly used to investigate samples of different conditions, e.g., patients versus controls. Systemic functional analyses emerged as a reference method to go beyond a list of regulated compounds, and identify activated or inactivated biological functions. This approach holds the promise for a better understanding of biological systems, of the mechanisms involved in disease progression, and thus improved diagnosis, prognosis, and treatment. In this chapter, we present a simple workflow to conduct pathway analyses on biological data using the freely available Reactome platform ( http://www.reactome.org ). PMID:26700057

  11. Equipment design issues for the in vivo X-ray fluorescence analysis of bone lead.

    PubMed Central

    Thomas, B J

    1991-01-01

    Several groups have reported the development of systems, based on the principle of X-ray fluorescence, for the in vivo measurement of bone lead concentrations. These systems have used the detection of either the characteristic L or K X-rays resulting from excitation by a suitable photon source. This paper examines design issues related to the development of these systems. These design issues are, in most instances, a result of consideration of the physical principles involved, and hence there are many features common to the systems developed by the individual groups. Design issues discussed in this paper include the selection of the site for measurement, source-sample-detector configuration, and collimation. Specific examples from published work are used to demonstrate the relevant features. PMID:2040249

  12. Analysis of Circulating MicroRNAs In Vivo following Administration of Dexamethasone and Adrenocorticotropin

    PubMed Central

    Igaz, Ivan; Nyír?, Gábor; Nagy, Zoltán; Butz, Henriett; Nagy, Zsolt; Perge, Pál; Sahin, Peter; Tóth, Miklós; Rácz, Károly; Igaz, Peter; Patócs, Attila

    2015-01-01

    Purpose. The interaction of hormones of the pituitary-adrenal axis and adrenal cortex-associated circulating microRNAs is mostly unknown. We have studied the effects of dexamethasone and adrenocorticotropin on the expression of five circulating microRNAs (hsa-miR-27a, hsa-miR-200b, hsa-miR-214, hsa-miR-483-5p, and hsa-miR-503) reported to be related to the adrenal cortex in plasma samples. Methods. Expression of microRNAs was studied in plasma samples of 10 individuals examined by 1?mg dexamethasone suppression test and another 10 individuals stimulated by 250??g tetracosactide (adrenocorticotropin). Total RNA was isolated and microRNA expression was analyzed by real-time reverse transcription quantitative polymerase chain reaction normalized to cel-miR-39 as reference. Results. Only circulating hsa-miR-27a proved to be significantly modulated in vivo by hormonal treatments: its expression was upregulated by dexamethasone whereas it was suppressed by adrenocorticotropin. Secreted hsa-miR-27a was significantly induced by dexamethasone in vitro in NCI-H295R cells, as well. The expression of hsa-miR-483-5p proposed as diagnostic marker for adrenocortical malignancy was not affected by dexamethasone or tetracosactide administration. Conclusions. hsa-miR-27a expression is modulated by hormones of the hypothalamic-pituitary-adrenal axis both in vitro and in vivo. The biological relevance of hsa-miR-27a modulation by hormones is unclear, but the responsiveness of circulating microRNAs to hormones of the pituitary-adrenal axis is noteworthy. PMID:26161091

  13. RNA Interference for Wheat Functional Gene Analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference (RNAi) refers to a common mechanism of RNA-based post-transcriptional gene silencing in eukaryotic cells. In model plant species such as Arabidopsis and rice, RNAi has been routinely used to characterize gene function and to engineer novel phenotypes. In polyploid species, this appr...

  14. Electromechanical Battery, Electrical Machines Mass Functions Analysis

    NASA Astrophysics Data System (ADS)

    Podgornovs, Andrejs; Sipovics, Anton

    2011-01-01

    In this paper different types of electrical machines in electromechanical battery, were described. The most known manufactured battery data is composed. Three types of machines: synchronous machine with salient poles and electromagnetic excitation, with permanent magnets on rotor and reluctance synchronous machine were analyzed. For all types of machines, mass is function of general geometrical size of magnetic system and machines electrical power.

  15. Genetic interaction analysis of point mutations enables interrogation of gene function at a residue-level resolution

    PubMed Central

    Braberg, Hannes; Moehle, Erica A.; Shales, Michael; Guthrie, Christine; Krogan, Nevan J.

    2014-01-01

    We have achieved a residue-level resolution of genetic interaction mapping – a technique that measures how the function of one gene is affected by the alteration of a second gene – by analyzing point mutations. Here, we describe how to interpret point mutant genetic interactions, and outline key applications for the approach, including interrogation of protein interaction interfaces and active sites, and examination of post-translational modifications. Genetic interaction analysis has proven effective for characterizing cellular processes; however, to date, systematic high-throughput genetic interaction screens have relied on gene deletions or knockdowns, which limits the resolution of gene function analysis and poses problems for multifunctional genes. Our point mutant approach addresses these issues, and further provides a tool for in vivo structure-function analysis that complements traditional biophysical methods. We also discuss the potential for genetic interaction mapping of point mutations in human cells and its application to personalized medicine. PMID:24842270

  16. Computational analysis, design, and experimental validation of antibody binding affinity improvements beyond in vivo maturation

    E-print Network

    Lippow, Shaun Matthew

    2007-01-01

    This thesis presents novel methods for the analysis and design of high-affinity protein interactions using a combination of high-resolution structural data and physics-based molecular models. First, computational analysis ...

  17. Analysis of the penetration of a caffeine containing shampoo into the hair follicles by in vivo laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Lademann, J.; Richter, H.; Schanzer, S.; Klenk, A.; Sterry, W.; Patzelt, A.

    2010-02-01

    In previous in vitro investigations, it was demonstrated that caffeine is able to stimulate the hair growth. Therefore, a penetration of caffeine into the hair follicle is necessary. In the present study, in vivo laser scanning microscopy (LSM) was used to investigate the penetration and storage of a caffeine containing shampoo into the hair follicles. It was shown that a 2-min contact time of the shampoo with the skin was enough to accumulate significant parts of the shampoo in the hair follicles. A penetration of the shampoo up to a depth of approx. 200 ?m could be detected, which represents the detection limit of the LSM. At this depth, the close network of the blood capillaries surrounding the hair follicles commences. Even after 24 h, the substance was still detectable in the hair follicles. This demonstrates the long-term reservoir function of the hair follicles for topically applied substances such as caffeine.

  18. Lab-on-a-brain: Implantable micro-optical fluidic devices for neural cell analysis in vivo

    NASA Astrophysics Data System (ADS)

    Takehara, Hiroaki; Nagaoka, Akira; Noguchi, Jun; Akagi, Takanori; Kasai, Haruo; Ichiki, Takanori

    2014-10-01

    The high-resolution imaging of neural cells in vivo has brought about great progress in neuroscience research. Here, we report a novel experimental platform, where the intact brain of a living mouse can be studied with the aid of a surgically implanted micro-optical fluidic device; acting as an interface between neurons and the outer world. The newly developed device provides the functions required for the long-term and high-resolution observation of the fine structures of neurons by two-photon laser scanning microscopy and the microfluidic delivery of chemicals or drugs directly into the brain. A proof-of-concept experiment of single-synapse stimulation by two-photon uncaging of caged glutamate and observation of dendritic spine shrinkage over subsequent days demonstrated a promising use for the present technology.

  19. Lab-on-a-brain: Implantable micro-optical fluidic devices for neural cell analysis in vivo

    PubMed Central

    Takehara, Hiroaki; Nagaoka, Akira; Noguchi, Jun; Akagi, Takanori; Kasai, Haruo; Ichiki, Takanori

    2014-01-01

    The high-resolution imaging of neural cells in vivo has brought about great progress in neuroscience research. Here, we report a novel experimental platform, where the intact brain of a living mouse can be studied with the aid of a surgically implanted micro-optical fluidic device; acting as an interface between neurons and the outer world. The newly developed device provides the functions required for the long-term and high-resolution observation of the fine structures of neurons by two-photon laser scanning microscopy and the microfluidic delivery of chemicals or drugs directly into the brain. A proof-of-concept experiment of single-synapse stimulation by two-photon uncaging of caged glutamate and observation of dendritic spine shrinkage over subsequent days demonstrated a promising use for the present technology. PMID:25335545

  20. Relation of myocardial oxygen consumption and function to high energy phosphate utilization during graded hypoxia and reoxygenation in sheep in vivo.

    PubMed Central

    Portman, M A; Standaert, T A; Ning, X H

    1995-01-01

    This study investigates the relation between myocardial oxygen consumption (MVO2), function, and high energy phosphates during severe hypoxia and reoxygenation in sheep in vivo. Graded hypoxia was performed in open-chested sheep to adjust PO2 to values where rapid depletion of energy stores occurred. Highly time-resolved 31P nuclear magnetic resonance spectroscopy enabled monitoring of myocardial phosphates throughout hypoxia and recovery with simultaneous MVO2 measurement. Sheep undergoing graded hypoxia (n = 5) with an arterial PO2 nadir of 13.4 +/- 0.5 mmHg, demonstrated maintained rates of oxygen consumption with large changes in coronary flow as phosphocreatine (PCr) decreased within 4 min to 40 +/- 7% of baseline. ATP utilization rate increased simultaneously 59 +/- 20%. Recovery was accompanied by marked increases in MVO2 from 2.0 +/- 0.5 to 7.2 +/- 1.9 mumol/g per min, while PCr recovery rate was 4.3 +/- 0.6 mumol/g per min. ATP decreased to 75 +/- 6% of baseline during severe hypoxia and did not recover. Sheep (n = 5) which underwent moderate hypoxia (PO2 maintained 25-35 mmHg for 10 min) did not demonstrate change in PCr or ATP. Functional and work assessment (n = 4) revealed that cardiac power increased during the graded hypoxia and was maintained through early reoxygenation. These studies show that (a) MVO2 does not decrease during oxygen deprivation in vivo despite marked and rapid decreases in high energy phosphates; (b) contractile function during hypoxia in vivo does not decrease during periods of PCr depletion and intracellular phosphate accumulation, and this may be related to marked increases in circulating catecholamines during global hypoxia. The measured creatine rephosphorylation rate is 34 +/- 11% of predicted (P < 0.01) calculated from reoxygenation parameters, which indicates that some mitochondrial respiratory uncoupling also occurs during the rephosphorylation period. Images PMID:7738181

  1. Intracellular cleavable poly(2-dimethylaminoethyl methacrylate) functionalized mesoporous silica nanoparticles for efficient siRNA delivery in vitro and in vivo.

    PubMed

    Lin, Daoshu; Cheng, Qiang; Jiang, Qian; Huang, Yuanyu; Yang, Zheng; Han, Shangcong; Zhao, Yuning; Guo, Shutao; Liang, Zicai; Dong, Anjie

    2013-05-21

    A low cytotoxicity and high efficiency delivery system with the advantages of low cost and facile fabrication is needed for the application of small interfering RNA (siRNA) delivery both in vitro and in vivo. For these prerequisites, cationic polymer-mesoporous silica nanoparticles (ssCP-MSNs) were prepared by surface functionalized mesoporous silica nanoparticles with disulfide bond cross-linked poly(2-dimethylaminoethyl methacrylate) (PDMAEMA). In vitro and in vivo evaluations were performed. The synthesized ssCP-MSNs are 100-150 nm in diameter with a pore size of 10 nm and a positively charged surface with a high zeta potential of 27 mV. Consequently, the ssCP-MSNs showed an excellent binding capacity for siRNA, and an enhancement in the cell uptake and cytosolic availability of siRNA. Furthermore, the intracellular reducing cleavage of the disulfide bonds cross-linking the PDMAEMA segments led to intracellular cleavage of PDMAEMA from ssCP-MSNs, which facilitated the intracellular triggered release of siRNA. Therefore, promoted RNA interference was observed in HeLa-Luc cells, which was equal to that of Lipofectamine 2000. Significantly, compared to Lipofectamine 2000, the ssCP-MSNs were more biocompatible, with low cytotoxicity (even non-cytotoxicity) and promotion of cell proliferation to HeLa-Luc cells. The in vivo systemic distribution studies certified that ssCP-MSNs/siRNA could prolong the duration of siRNA in vivo, and that they accumulated in the adrenal gland, liver, lung, spleen, kidney, heart and thymus after intravenous injection. Encouragingly, with the ability to deliver siRNA to a tumor, ssCP-MSNs/siRNA showed a tumor suppression effect in the HeLa-Luc xenograft murine model after intravenous injection. Therefore, the ssCP-MSNs cationic polymer-mesoporous silica nanoparticles with low cytotoxicity are promising for siRNA delivery. PMID:23552843

  2. DISSIMILARITY-BASED FUNCTIONS FOR ECOLOGICAL ANALYSIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The package ecodistR is an add-on developed for R statistical software (R Foundation for Statistical Computing, Vienna, Austria). It implements statistical analysis methods developed by the authors and others for use with ecological data. The main focus is on multivariate, spatial and dissimilarity...

  3. In vivo measurement of the shape of the tissue-refractive-index correlation function and its application to detection of colorectal field carcinogenesis.

    PubMed

    Gomes, Andrew J; Ruderman, Sarah; DelaCruz, Mart; Wali, Ramesh K; Roy, Hemant K; Backman, Vadim

    2012-04-01

    Polarization-gated spectroscopy is an established method to depth-selectively interrogate the structural properties of biological tissue. We employ this method in vivo in the azoxymethane (AOM)-treated rat model to monitor the morphological changes that occur in the field of a tumor during early carcinogenesis. The results demonstrate a statistically significant change in the shape of the refractive-index correlation function for AOM-treated rats versus saline-treated controls. Since refractive index is linearly proportional to mass density, these refractive-index changes can be directly linked to alterations in the spatial distribution patterns of macromolecular density. Furthermore, we found that alterations in the shape of the refractive-index correlation function shape were an indicator of both present and future risk of tumor development. These results suggest that noninvasive measurement of the shape of the refractive-index correlation function could be a promising marker of early cancer development. PMID:22559696

  4. A conserved salt bridge in the G loop of multiple protein kinases is important for catalysis and for in vivo Lyn function.

    PubMed

    Barouch-Bentov, Rina; Che, Jianwei; Lee, Christian C; Yang, Yating; Herman, Ann; Jia, Yong; Velentza, Anastasia; Watson, James; Sternberg, Luise; Kim, Sunjun; Ziaee, Niusha; Miller, Andrew; Jackson, Carie; Fujimoto, Manabu; Young, Mike; Batalov, Serge; Liu, Yi; Warmuth, Markus; Wiltshire, Tim; Cooke, Michael P; Sauer, Karsten

    2009-01-16

    The glycine-rich G loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases, from plants to humans. By limiting G loop flexibility, it controls ATP binding, catalysis, and inhibition by ATP-competitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn(-/-) mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein. PMID:19150426

  5. In vivo measurement of the shape of the tissue-refractive-index correlation function and its applicationto detection of colorectal field carcinogenesis

    NASA Astrophysics Data System (ADS)

    Gomes, Andrew J.; Ruderman, Sarah; DelaCruz, Mart; Wali, Ramesh K.; Roy, Hemant K.; Backman, Vadim

    2012-04-01

    Polarization-gated spectroscopy is an established method to depth-selectively interrogate the structural properties of biological tissue. We employ this method in vivo in the azoxymethane (AOM)-treated rat model to monitor the morphological changes that occur in the field of a tumor during early carcinogenesis. The results demonstrate a statistically significant change in the shape of the refractive-index correlation function for AOM-treated rats versus saline-treated controls. Since refractive index is linearly proportional to mass density, these refractive-index changes can be directly linked to alterations in the spatial distribution patterns of macromolecular density. Furthermore, we found that alterations in the shape of the refractive-index correlation function shape were an indicator of both present and future risk of tumor development. These results suggest that noninvasive measurement of the shape of the refractive-index correlation function could be a promising marker of early cancer development.

  6. Functional Foods Baseline and Requirements Analysis

    NASA Technical Reports Server (NTRS)

    Cooper, M. R.; Bermudez-Aguirre, L. D.; Douglas, G.

    2015-01-01

    Current spaceflight foods were evaluated to determine if their nutrient profile supports positioning as a functional food and if the stability of the bioactive compound within the food matrix over an extended shelf-life correlated with the expected storage duration during the mission. Specifically, the research aims were: Aim A. To determine the amount of each nutrient in representative spaceflight foods immediately after processing and at predetermined storage time to establish the current nutritional state. Aim B. To identify the requirements to develop foods that stabilize these nutrients such that required concentrations are maintained in the space food system throughout long duration missions (up to five years). Aim C. To coordinate collaborations with health and performance groups that may require functional foods as a countermeasure.

  7. [Coupling of membranous and metabolic functions in nucleated erythrocytes of Scorpaena porcus L. in hypoxia (experiments in vivo and in vitro)].

    PubMed

    Soldatov, A A; Andreeva, A Yu; Novitskaya, V N; Parfenova, I A

    2014-01-01

    Effect of hypoxia (diapason of 0.57-8.17 mg O2 l(-1)) on functional characteristics of nucleated erythrocytes of the benthonic marine fish Scorpaena porcus L. was studied under conditions in vivo and in vitro. It has been established that characteristic of cellular system of this species is a balanced unhibition of membranous and metabolic functions under conditions of external deficit of oxygen (experiments in vivo). This is expressed in the ability of cells to maintain within norm the intracellular ATP concentration and transmembrane gradients for Na+ and K+ with low activities of Na+, K(+)-ATPase and hexokinase. This phenomenon seems to be based on the process of a decrease of the number of functioning ion channel at the level of the cell cytoplasmic membrane; this process reduces energy expenditure for maintenance of cationic gradients (the phenomenon of metabolic arrest). The same is also indicated by an increase of intensity of fluorescence of FDA and R123 in the scorpaena erythrocytic suspensions in hypoxia (experiments in vitro). Mechanisms underlying this phenomenon are discussed. PMID:25786318

  8. In vivo direct reprogramming of reactive glial cells into functional neurons after brain injury and in an Alzheimer’s disease model

    PubMed Central

    Guo, Ziyuan; Zhang, Lei; Wu, Zheng; Chen, Yuchen; Wang, Fan; Chen, Gong

    2014-01-01

    Summary Loss of neurons after brain injury and in neurodegenerative disease is often accompanied by reactive gliosis and scarring, which are difficult to reverse with existing treatment approaches. Here, we show that reactive glial cells in the cortex of stab-injured or Alzheimer’s disease (AD) model mice can be directly reprogrammed into functional neurons in vivo using retroviral expression of a single neural transcription factor, NeuroD1. Following expression of NeuroD1, astrocytes were reprogrammed into glutamatergic neurons, while NG2 cells were reprogrammed into glutamatergic and GABAergic neurons. Cortical slice recordings revealed both spontaneous and evoked synaptic responses in NeuroD1-converted neurons, suggesting that they integrated into local neural circuits. NeuroD1 expression was also able to reprogram cultured human cortical astrocytes into functional neurons. Our studies therefore suggest that direct reprogramming of reactive glial cells into functional neurons in vivo could provide an alternative approach for repair of injured or diseased brain. PMID:24360883

  9. Functional analysis of the Fusarium graminearum phosphatome.

    PubMed

    Yun, Yingzi; Liu, Zunyong; Yin, Yanni; Jiang, Jinhua; Chen, Yun; Xu, Jin-Rong; Ma, Zhonghua

    2015-07-01

    Phosphatases are known to play important roles in the regulation of various cellular processes in eukaryotes. However, systematic characterization of the phosphatome has not been reported in phytopathogenic fungi. The wheat scab fungus Fusarium graminearum contains 82 putative phosphatases. The biological functions of each phosphatase were investigated in this study. Although 11 phosphatase genes appeared to be essential, deletion mutants of the other 71 phosphatase genes were obtained and characterized for changes in 15 phenotypes, including vegetative growth, nutrient response and virulence. Overall, the deletion of 63 phosphatase genes resulted in changes in at least one of the phenotypes assayed. Interestingly, the deletion of four genes (Fg06297, Fg03333, Fg03826 and Fg07932) did not dramatically affect hyphal growth, but led to strongly reduced virulence. Western blot analyses showed that three phosphatases (Fg10516, Fg03333 and Fg12867) functioned as negative regulators of the mitogen-activated protein kinase signaling pathways. In addition, we found, for the first time, that FgCdc14 is dispensable for growth, but plays an important role in ribosome biogenesis. Overall, in this first functional characterization of the fungal phosphatome, phosphatases important for various aspects of hyphal growth, development, plant infection and secondary metabolism were identified in the phytopathogenic fungus F. graminearum. PMID:25758923

  10. Use of D-T-produced fast neutrons for in vivo body composition analysis: a reference method for nutritional assessment in the elderly.

    PubMed

    Kehayias, J J

    2004-05-01

    Body composition has become the main outcome of many nutritional intervention studies including osteoporosis, malnutrition, obesity, AIDS, and aging. Traditional indirect body composition methods developed with healthy young adults do not apply to the elderly or diseased. Fast neutron activation (for N and P) and neutron inelastic scattering (for C and O) are used to assess in vivo elements characteristic of specific body compartments. Non-bone phosphorus for muscle is measured by the (31)P(n, alpha)(28)Al reaction, and nitrogen for protein via the (14)N(n,2n)(13)N fast neutron reaction. Inelastic neutron scattering is used to measure total body carbon and oxygen. Body fat is derived from carbon after correcting for contributions from protein, bone, and glycogen. Carbon-to-oxygen ratio (C/O) is used to measure the distribution of fat and lean tissue in the body and to monitor small changes of lean mass. A sealed, D-T neutron generator is used for the production of fast neutrons. Carbon and oxygen mass and their ratio are measured in vivo at a radiation exposure of less than 0.06 mSv. Gamma-ray spectra are collected using large BGO detectors and analyzed for the 4.43 MeV state of carbon and 6.13 MeV state of oxygen, simultaneously with the irradiation. P and N analysis by delayed fast neutron activation is performed by transferring the patient to a shielded room equipped with an array of NaI(Tl) detectors. A combination of measurements makes possible the assessment of the "quality" of fat-free mass. The neutron generator system is used to evaluate the efficacy of new treatments, to study mechanisms of lean tissue depletion with aging, and to investigate methods for preserving function and quality of life in the elderly. It is also used as a reference method for the validation of portable instruments of nutritional assessment. PMID:14747891

  11. Technological Barriers in the Use of Electrochemical Microsensors and Microbiosensors for in vivo Analysis of Neurological Relevant Substances

    PubMed Central

    Bucur, Bogdan

    2012-01-01

    In this paper is presented an overview of the technological barriers faced by the in vivo brain analysis with microelectrodes. Numerous microsensors and enzymatic microbiosensors have been developed for the real time monitoring of neurotransmitters, neuromodulators, drugs and diverse other biological relevant substances. A clear understanding of the working principle, advantages and limitations is essential for the acquisition of valid data in neurological investigations. Some of the aspects presented here refer to: microelectrode insertion and positioning related to possibilities to minimize tissue damage, spatial and temporal resolution of the measurements, actual controversies in data interpretation and sensor calibration, simultaneous detection of multiple analytes, interferences and state of the art in the development of wireless devices. PMID:23449399

  12. Establishment of the forward genetic analysis of the chlorophyll d-dominated cyanobacterium Acaryochloris marina MBIC 11017 by applying in vivo transposon mutagenesis system.

    PubMed

    Watabe, Kazuyuki; Mimuro, Mamoru; Tsuchiya, Tohru

    2015-08-01

    Acaryochloris marina MBIC 11017 possesses chlorophyll (Chl) d as a major Chl, which enables this organism to utilize far-red light for photosynthesis. Thus, the adaptation mechanism of far-red light utilization, including Chl d biosynthesis, has received much attention, though a limited number of reports on this subject have been published. To identify genes responsible for Chl d biosynthesis and adaptation to far-red light, molecular genetic analysis of A. marina was required. We developed a transformation system for A. marina and introduced expression vectors into A. marina. In this study, the high-frequency in vivo transposon mutagenesis system recently established by us was applied to A. marina. As a result, we obtained mutants with the transposon in their genomic DNA at various positions. By screening transposon-tagged mutants, we isolated a mutant (Y1 mutant) that formed a yellow colony on agar medium. In the Y1 mutant, the transposon was inserted into the gene encoding molybdenum cofactor biosynthesis protein A (MoaA). The Y1 mutant was functionally complemented by introducing the moaA gene or increasing the ammonium ion in the medium. These results indicate that the mutation of the moaA gene reduced nitrate reductase activity, which requires molybdenum cofactor, in the Y1 mutant. This is the first successful forward genetic analysis of A. marina, which will lead to the identification of genes responsible for adaptation to far-red light. PMID:25596846

  13. Targeted Resequencing and Systematic In Vivo Functional Testing Identifies Rare Variants in MEIS1 as Significant Contributors to Restless Legs Syndrome

    PubMed Central

    Schulte, Eva C.; Kousi, Maria; Tan, Perciliz L.; Tilch, Erik; Knauf, Franziska; Lichtner, Peter; Trenkwalder, Claudia; Högl, Birgit; Frauscher, Birgit; Berger, Klaus; Fietze, Ingo; Hornyak, Magdolna; Oertel, Wolfgang H.; Bachmann, Cornelius G.; Zimprich, Alexander; Peters, Annette; Gieger, Christian; Meitinger, Thomas; Müller-Myhsok, Bertram; Katsanis, Nicholas; Winkelmann, Juliane

    2014-01-01

    Restless legs syndrome (RLS) is a common neurologic condition characterized by nocturnal dysesthesias and an urge to move, affecting the legs. RLS is a complex trait, for which genome-wide association studies (GWASs) have identified common susceptibility alleles of modest (OR 1.2–1.7) risk at six genomic loci. Among these, variants in MEIS1 have emerged as the largest risk factors for RLS, suggesting that perturbations in this transcription factor might be causally related to RLS susceptibility. To establish this causality, direction of effect, and total genetic burden of MEIS1, we interrogated 188 case subjects and 182 control subjects for rare alleles not captured by previous GWASs, followed by genotyping of ?3,000 case subjects and 3,000 control subjects, and concluded with systematic functionalization of all discovered variants using a previously established in vivo model of neurogenesis. We observed a significant excess of rare MEIS1 variants in individuals with RLS. Subsequent assessment of all nonsynonymous variants by in vivo complementation revealed an excess of loss-of-function alleles in individuals with RLS. Strikingly, these alleles compromised the function of the canonical MEIS1 splice isoform but were irrelevant to an isoform known to utilize an alternative 3? sequence. Our data link MEIS1 loss of function to the etiopathology of RLS, highlight how combined sequencing and systematic functional annotation of rare variation at GWAS loci can detect risk burden, and offer a plausible explanation for the specificity of phenotypic expressivity of loss-of-function alleles at a locus broadly necessary for neurogenesis and neurodevelopment. PMID:24995868

  14. Clearance and early hydrolysis of atrial natriuretic factor in vivo. Structural analysis of cleavage sites and design of an analogue that inhibits hormone cleavage.

    PubMed Central

    Condra, C L; Leidy, E A; Bunting, P; Colton, C D; Nutt, R F; Rosenblatt, M; Jacobs, J W

    1988-01-01

    This study examines the clearance and early hydrolysis of atrial natriuretic factor (ANF) in vivo. Radiolabeled ANF was cleared from the circulation of the rat with biphasic kinetics; the majority (90%) of ANF cleared with a t1/2 of 15 s, the remaining peptide was cleared with a t1/2 of 5 min. Microsequence analysis of ANF peptides recovered from the circulation of rats revealed five major degradation products of the intact hormone. The first cleavage occurred between amino acids 12 and 13 of the hormone and would inactivate ANF. Over time, additional fragments of the hormone were generated, including fragments of 6, 7, 21, and 24 amino acids in length. Whole body radioautography of rats injected with [123I]-ANF revealed the kidney as a predominant organ involved in clearance of ANF. Subsequent amino acid sequence analyses of radiolabeled ANF exposed to the kidney in vivo indicated that this organ generated four of the five major hydrolysis products observed in circulation, namely, the 6, 7, 16, and 21 amino acid fragments of the hormone. In an attempt to stabilize ANF in vivo, a synthetic analogue of the hormone was prepared that contained the amino acid analogue, aminoisobutyric acid, substituted at position 13. This analogue completely abolished the in vivo cleavage of ANF at this site. These studies demonstrate the usefulness of a protein chemistry approach in characterizing hormone metabolism in vivo and designing analogues with enhanced in vivo stability to cleavage. Images PMID:2966813

  15. Analysis of in vitro and in vivo effects of probiotics against Campylobacter spp.

    PubMed

    Bratz, Katharina; Gölz, Greta; Janczyk, Pawel; Nöckler, Karsten; Alter, Thomas

    2015-01-01

    Campylobacter (C.) spp. are well recognised as the leading cause of bacterial food-borne diarrheal disease worldwide, with C. jejuni and C. coli as the most important species: C. coli is highly abundant in pigs and pork meat has often been implicated as a source for human infection. Intestinal colonisation of C. coli in pigs plays a role in carcass contamination during slaughter. Different pre-harvest intervention measures are proposed to reduce the C. coli burden in the porcine intestine. Among others, the use of probiotics to prevent or reduce the colonisation of intestinal pathogens is discussed. One aim of this study was to screen a variety of probiotics to evaluate their inhibitory activity against Campylobacter spp. in vitro. Therefore, cell-free culture supernatants of Lactobacillus spp., Bifidobacterium spp., Enterococcus (E.) faecium NCIMB 10415, and Escherichia coli Nissle 1917 were tested against C. jejuni and C. coli by a well-diffusion agar assay. Seven out of eleven Lactobacillus strains showed an inhibitory activity against at least one of the three tested Campylobacter strains. This antagonistic activity against Campylobacter spp. was caused by the production of organic acids that lowered the pH. Application with pH neutralised cell-free culture supernatants abolished this inhibitory effect. Other tested strains with probiotic properties showed no inhibitory activity against any Campylobacter spp. strain. The strain E. faecium NCIMB 10415 was chosen to test its inhibitory activity against C. coli in vivo. Twenty weaned piglets were allocated into two groups, a probiotic group and a control group.The diet of the probiotic group was supplemented with E. faecium NCIMB 10415 (10(9) cfu/kg feed, Cylactin) since weaning, whereas the control group received no probiotic treatment. All piglets were naturally colonised with C. coli. The excretion load of C. coli was monitored for 28 days. The results indicate that dietary supplementation of E. faecium NCIMB 10415 did not significantly affect C. coli excretion levels in pigs. In this study, E. faecium NCIMB 10415 showed no antagonistic activity against C. coli in vitro and in vivo and had no impact on the growth performance of weaned piglets. PMID:25876276

  16. Functional imaging of auditory scene analysis.

    PubMed

    Gutschalk, Alexander; Dykstra, Andrew R

    2014-01-01

    Our auditory system is constantly faced with the task of decomposing the complex mixture of sound arriving at the ears into perceptually independent streams constituting accurate representations of individual sound sources. This decomposition, termed auditory scene analysis, is critical for both survival and communication, and is thought to underlie both speech and music perception. The neural underpinnings of auditory scene analysis have been studied utilizing invasive experiments with animal models as well as non-invasive (MEG, EEG, and fMRI) and invasive (intracranial EEG) studies conducted with human listeners. The present article reviews human neurophysiological research investigating the neural basis of auditory scene analysis, with emphasis on two classical paradigms termed streaming and informational masking. Other paradigms - such as the continuity illusion, mistuned harmonics, and multi-speaker environments - are briefly addressed thereafter. We conclude by discussing the emerging evidence for the role of auditory cortex in remapping incoming acoustic signals into a perceptual representation of auditory streams, which are then available for selective attention and further conscious processing. This article is part of a Special Issue entitled Human Auditory Neuroimaging. PMID:23968821

  17. JOURNAL OF THE EXPERIMENTAL ANALYSIS OF BEHAVIOR A FUNCTIONAL ANALYSIS OF LANGUAGE'

    E-print Network

    Premack, David

    form of syntax and phonology, upon which the basic functions of language depend. Strict Training alternative ways of producing the function. A strict training pro- cedure is'essentially a recipe. GivenJOURNAL OF THE EXPERIMENTAL ANALYSIS OF BEHAVIOR A FUNCTIONAL ANALYSIS OF LANGUAGE' DAVID PREMACK

  18. ASYMPTOTIC ANALYSIS OF A CLASS OF FUNCTIONAL EQUATIONS AND APPLICATIONS

    E-print Network

    Prodinger, Helmut

    --Hungarian Science Cooperation Program, project 10U3 Typeset by A M S­T E X 1 #12; 2 PETER J. GRABNER, HELMUTASYMPTOTIC ANALYSIS OF A CLASS OF FUNCTIONAL EQUATIONS AND APPLICATIONS Peter J. Grabnery, Helmut

  19. ORIGINAL PAPER Analysis of subcellular surface structure, function

    E-print Network

    Bielefeld, Universität

    , the subcellular surface structure of living bacteria (Corynebacterium glutamicum) was investigated with atomic Single cell analysis at the subcellular level gives quantitative information from a structural mechanisms of specific interaction, binding kinetics and the interplay of genomic information and functional

  20. Grammatical Analysis as a Distributed Neurobiological Function

    PubMed Central

    Bozic, Mirjana; Fonteneau, Elisabeth; Su, Li; Marslen-Wilson, William D

    2015-01-01

    Language processing engages large-scale functional networks in both hemispheres. Although it is widely accepted that left perisylvian regions have a key role in supporting complex grammatical computations, patient data suggest that some aspects of grammatical processing could be supported bilaterally. We investigated the distribution and the nature of grammatical computations across language processing networks by comparing two types of combinatorial grammatical sequences—inflectionally complex words and minimal phrases—and contrasting them with grammatically simple words. Novel multivariate analyses revealed that they engage a coalition of separable subsystems: inflected forms triggered left-lateralized activation, dissociable into dorsal processes supporting morphophonological parsing and ventral, lexically driven morphosyntactic processes. In contrast, simple phrases activated a consistently bilateral pattern of temporal regions, overlapping with inflectional activations in L middle temporal gyrus. These data confirm the role of the left-lateralized frontotemporal network in supporting complex grammatical computations. Critically, they also point to the capacity of bilateral temporal regions to support simple, linear grammatical computations. This is consistent with a dual neurobiological framework where phylogenetically older bihemispheric systems form part of the network that supports language function in the modern human, and where significant capacities for language comprehension remain intact even following severe left hemisphere damage. PMID:25421880

  1. Immunochemical analysis of kinesin light chain function.

    PubMed Central

    Stenoien, D L; Brady, S T

    1997-01-01

    The kinesin heterotetramer consists of two heavy and two light chains. Kinesin light chains have been proposed to act in binding motor protein to cargo, but evidence for this has been indirect. A library of monoclonal antibodies directed against conserved epitopes throughout the kinesin light chain sequence were used to map light chain functional architecture and to assess physiological functions of these domains. Immunocytochemistry with all antibodies showed a punctate pattern that was detergent soluble. A monoclonal antibody (KLC-All) made against a highly conserved epitope in the tandem repeat domain of light chains inhibited fast axonal transport in isolated axoplasm by decreasing both the number and velocity of vesicles moving, whereas an antibody against a conserved amino terminus epitope had no effect. KLC-All was equally effective at inhibiting both anterograde and retrograde transport. Neither antibody inhibited microtubule-binding or ATPase activity in vitro. KLC-All was unique among antibodies tested in releasing kinesin from purified membrane vesicles, suggesting a mechanism of action for inhibition of axonal transport. These results provide further evidence that conventional kinesin is a motor for fast axonal transport and demonstrate that kinesin light chains play an important role in kinesin interaction with membranes. Images PMID:9247647

  2. Analysis of uranium urinalysis and in vivo measurement results from eleven participating uranium mills

    SciTech Connect

    Spitz, H.B.; Simpson, J.C.; Aldridge, T.L.

    1984-05-01

    Uranium urinalysis and in vivo examination results obtained from workers at eleven uranium mills between 1978 and 1980 were evaluated. The main purpose was to determine the degree of the mills' compliance with bioassay monitoring recommendations given in the draft NRC Regulatory Guide 8.22 (USNRC 1978). The effect of anticipated changes in the draft regulatory guidance, as expressed to PNL in May 1982, was also studied. Statistical analyses of the data showed that the bioassay results did not reliably meet the limited performance criteria given in the draft regulatory guide. Furthermore, quality control measurements of uranium in urine indicated that detection limits at ..cap alpha.. = ..beta.. = 0.05 ranged from 13 ..mu..g/l to 29 ..mu..g/l, whereas the draft regulatory guidance suggests 5 ..mu..g/l as the detection limit. Recommendations for monitoring frequencies given in the draft guide were not followed consistently from mill to mill. The results of these statistical analyses indicate a need to include performance criteria for accuracy, precision, and confidence in revisions of the draft Regulatory Guide 8.22. Revised guidance should also emphasize the need for each mill to continually test the laboratory performing urinalyses by submitting quality control samples (i.e., blank and spiked urine samples as open and blind test) to insure that the performance criteria are being met. Recommendations for a bioassay audit program are also given. 25 references, 15 figures, 17 tables.

  3. New developments in two-photon analysis of human skin in vivo

    NASA Astrophysics Data System (ADS)

    Riemann, I.; Schwarz, M.; Stracke, F.; Ehlers, A.; Dimitrow, E.; Kaatz, M.; König, K.; Le Harzic, R.

    2009-02-01

    Two-photon imaging of human skin using ultra short laser pulses can be used to obtain information about the state of cells and tissues by means of their natural autofluorescence. Using this method, it is possible to determine whether the normal cell pattern is disturbed or the autofluorescence is influenced by internal or external stimuli. Two-photon fluorescence lifetime imaging (FLIM) can further enhance this providing information about physiological processes, fluorophores (like NAD(P)H, collagen, keratin, elastin, flavins, melanin,...) and external applied probes inside cells and tissue parts. For example the part of the cells metabolism and energy level can be determined by analyzing the NADH regarding its free / bound state and its oxidized / reduced state. The combination of two-photon imaging with FLIM may lead to a better understanding and diagnosis of skin reactions and disorders. We also present some results of in vivo simultaneous collagen and elastin measurements in skin dermis. Changes of dermal collagen and elastin content are characteristic for skin aging as well as for pathological skin conditions.

  4. First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate

    PubMed Central

    Dooley, Helen; Stanfield, Robyn L.; Brady, Rebecca A.; Flajnik, Martin F.

    2006-01-01

    The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (Ginglymostoma cirratum) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the in vivo maturation mechanisms, in general, and for single-domain antibody fragments. PMID:16446445

  5. Contact fiber probes for in-vivo optical spectroscopy: comparative analysis

    NASA Astrophysics Data System (ADS)

    Denisov, Nikolay A.; Griffin, Stephen E.

    1998-04-01

    In recent years, in-vivo optical spectroscopy has become widely used during many routine endoscopic procedures for purposes of biomedical science and practical medicine by virtue of its non-invasive effect and real time remote detection convenience. Fiber optic probes are an important unit of spectroscopic equipment for providing effective excitation and light gathering on site. Probes should be small enough to be easy introduced into the instrumentation channel of standard flexible or rigid endoscopes. Several commercial and custom types of fiber tips for applications in diagnostics have been examined. Single- and multifiber delivery schemes are also discussed. To provide comparative analyses of probes' optical properties, sensor optical efficiency and tip coupling efficiency coefficients have been proposed. These coefficients are a quantitative measure of probe optical efficiency and have been calculated by means of ray tracing for both fiber-tissue and tissue-fiber traces. These results could be a helpful tool for designers of fiber probes for Raman, laser-induced fluorescence and elastic-scattered spectroscopy of internal human organs.

  6. In?Vitro and In?Vivo Noise Analysis for Optical Neural Recording

    PubMed Central

    Foust, Amanda J.; Schei, Jennifer L.; Rojas, Manuel J.; Rector, David M.

    2008-01-01

    Laser diodes (LD) are commonly used for optical neural recordings in chronically recorded animals and humans, primarily due to their brightness and small size. However, noise introduced by LDs may counteract the benefits of brightness when compared to low?noise light emitting diodes (LEDs). To understand noise sources in optical recordings, we systematically compared instrument and physiological noise profiles in two recording paradigms. A better understanding of noise sources will help improve optical recordings and make them more practical with fewer averages. We stimulated lobster nerves and rat cortex, then compared the root mean square (RMS) noise and signal?to?noise ratios (SNRs) of data obtained with LED, superluminescent diode (SLD) and LD illumination for different numbers of averages. The LED data exhibited significantly higher SNRs in fewer averages than LD data in all recordings. In the absence of tissue, LED noise increased linearly with intensity, while LD noise increased sharply in the transition to lasing and settled to noise levels significantly higher than the LED’s, suggesting that speckle noise contributed to the LD’s higher noise and lower SNRs. Our data recommend low coherence and portable light sources for in?vivo chronic neural recording applications. PMID:19021365

  7. Detection of Islet ?-Cell Death in Vivo by Multiplex PCR Analysis of Differentially Methylated DNA

    PubMed Central

    Fisher, Marisa M.; Perez Chumbiauca, Cristina N.; Mather, Kieren J.; Mirmira, Raghavendra G.

    2013-01-01

    Noninvasive detection of early ?-cell death in type 1 diabetes might identify individuals in whom therapeutic interventions would preserve ?-cell mass and prevent hyperglycemia. Recent studies in mice have shown that ?-cell death produces a corresponding increase in unmethylated preproinsulin (PPI) DNA in serum. Here, we report the development of a novel assay using dual fluorescent-probe multiplex PCR (TaqMan) to detect differential methylation of circulating PPI DNA. Key assay features include low background signals, linear assay output across a large range of values, and simultaneous detection of methylated and unmethylated PPI DNA in a single reaction. We defined the “unmethylation index” as a summary parameter that reflects the relative amounts of unmethylated vs methylated PPI DNA. To validate this assay's ability to detect ?-cell death in vivo, we measured the unmethylation index in the serum of diabetic mouse models, including high- and multiple low-dose streptozotocin-induced diabetes, and the nonobese diabetic mouse model of type 1 diabetes. Our data show a significantly increased unmethylation index concordant with the known timeline of ?-cell death that precedes the onset of hyperglycemia. Subsequently, we observed a decrease in the unmethylation index following diabetes development, likely reflecting the absence of further ?-cell death in the pancreas. We conclude that simultaneous measurement of methylated and unmethylated PPI DNA using the multiplex PCR method described here is a readily available and sensitive indicator of dying ?-cells that may be useful to track diabetes progression and response to therapeutic intervention. PMID:23825129

  8. MDA-7/IL-24 functions as a tumor suppressor gene in vivo in transgenic mouse models of breast cancer.

    PubMed

    Menezes, Mitchell E; Shen, Xue-Ning; Das, Swadesh K; Emdad, Luni; Guo, Chunqing; Yuan, Fang; Li, You-Jun; Archer, Michael C; Zacksenhaus, Eldad; Windle, Jolene J; Subler, Mark A; Ben-David, Yaacov; Sarkar, Devanand; Wang, Xiang-Yang; Fisher, Paul B

    2015-11-10

    Melanoma differentiation associated gene-7/Interleukin-24 (MDA-7/IL-24) is a novel member of the IL-10 gene family that selectively induces apoptosis and toxic autophagy in a broad spectrum of human cancers, including breast cancer, without harming normal cells or tissues. The ability to investigate the critical events underlying cancer initiation and progression, as well as the capacity to test the efficacy of novel therapeutics, has been significantly advanced by the development of genetically engineered mice (GEMs) that accurately recapitulate specific human cancers. We utilized three transgenic mouse models to better comprehend the in vivo role of MDA-7/IL-24 in breast cancer. Using the MMTV-PyMT spontaneous mammary tumor model, we confirmed that exogenously introducing MDA-7/IL-24 using a Cancer Terminator Virus caused a reduction in tumor burden and also produced an antitumor "bystander" effect. Next we performed xenograft studies in a newly created MMTV-MDA-7 transgenic model that over-expresses MDA-7/IL-24 in the mammary glands during pregnancy and lactation, and found that MDA-7/IL-24 overexpression delayed tumor growth following orthotopic injection of a murine PDX tumor cell line (mPDX) derived from a tumor formed in an MMTV-PyMT mouse. We also crossed the MMTV-MDA-7 line to MMTV-Erbb2 transgenic mice and found that MDA-7/IL-24 overexpression delayed the onset of mammary tumor development in this model of spontaneous mammary tumorigenesis as well. Finally, we assessed the role of MDA-7/IL-24 in immune regulation, which can potentially contribute to tumor suppression in vivo. Our findings provide further direct in vivo evidence for the role of MDA-7/IL-24 in tumor suppression in breast cancer in immune-competent transgenic mice. PMID:26474456

  9. Formulation, High Throughput In Vitro Screening and In Vivo Functional Characterization of Nanoemulsion-Based Intranasal Vaccine Adjuvants

    PubMed Central

    Wong, Pamela T.; Leroueil, Pascale R.; Smith, Douglas M.; Ciotti, Susan; Bielinska, Anna U.; Janczak, Katarzyna W.; Mullen, Catherine H.; Groom, Jeffrey V.; Taylor, Erin M.; Passmore, Crystal; Makidon, Paul E.; O’Konek, Jessica J.; Myc, Andrzej; Hamouda, Tarek; Baker, James R.

    2015-01-01

    Vaccine adjuvants have been reported to induce both mucosal and systemic immunity when applied to mucosal surfaces and this dual response appears important for protection against certain pathogens. Despite the potential advantages, however, no mucosal adjuvants are currently approved for human use. Evaluating compounds as mucosal adjuvants is a slow and costly process due to the need for lengthy animal immunogenicity studies. We have constructed a library of 112 intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions that contain various cationic and nonionic surfactants. To facilitate adjuvant development we first evaluated this library in a series of high-throughput, in vitro assays for activities associated with innate and adaptive immune activation in vivo. These in vitro assays screened for the ability of the adjuvant to bind to mucin, induce cytotoxicity, facilitate antigen uptake in epithelial and dendritic cells, and activate cellular pathways. We then sought to determine how these parameters related to adjuvant activity in vivo. While the in vitro assays alone were not enough to predict the in vivo adjuvant activity completely, several interesting relationships were found with immune responses in mice. Furthermore, by varying the physicochemical properties of the surfactant components (charge, surfactant polar head size and hydrophobicity) and the surfactant blend ratio of the formulations, the strength and type of the immune response generated (TH1, TH2, TH17) could be modulated. These findings suggest the possibility of using high-throughput screens to aid in the design of custom adjuvants with unique immunological profiles to match specific mucosal vaccine applications. PMID:25962136

  10. Noninvasive analysis of human neck muscle function

    NASA Technical Reports Server (NTRS)

    Conley, M. S.; Meyer, R. A.; Bloomberg, J. J.; Feeback, D. L.; Dudley, G. A.

    1995-01-01

    STUDY DESIGN. Muscle use evoked by exercise was determined by quantifying shifts in signal relaxation times of T2-weighted magnetic resonance images. Images were collected at rest and after exercise at each of two intensities (moderate and intense) for each of four head movements: 1) extension, 2) flexion, 3) rotation, and 4) lateral flexion. OBJECTIVE. This study examined the intensity and pattern of neck muscle use evoked by various movements of the head. The results will help elucidate the pathophysiology, and thus methods for treating disorders of the cervical musculoskeletal system. SUMMARY OF BACKGROUND DATA. Exercise-induced contrast shifts in T2 has been shown to indicate muscle use during the activity. The noninvasive nature of magnetic resonance imaging appears to make it an ideal approach for studying the function of the complex neuromuscular system of the neck. METHODS. The extent of T2 increase was examined to gauge how intensely nine different neck muscles or muscle pairs were used in seven subjects. The absolute and relative cross-sectional area of muscle showing a shift in signal relaxation was assessed to infer the pattern of use among and within individual neck muscles or muscle pairs. RESULTS. Signal relaxation increased with exercise intensity for each head movement. The absolute and relative cross-sectional area of muscle showing a shift in signal relaxation also increased with exercise load. Neck muscles or muscle pairs extensively used to perform each head movement were: extension--semispinalis capitis and cervicis and splenius capitis; flexion--sternocleidomastoid and longus capitis and colli; rotation--splenius capitis, levator scapulae, scalenus, semispinalis capitis ipsilateral to the rotation, and sternocleidomastoid contralateral; and lateral flexion--sternocleidomastoid CONCLUSION. The results of this study, in part, agree with the purported functions of neck muscles derived from anatomic location. This also was true for the few selected muscles that have been examined in human electromyographic studies. Neck muscle function and morphology can be studied at a detailed level using exercise-induced shifts in magnetic resonance images.

  11. Insulin Resistance Is Not Associated with an Impaired Mitochondrial Function in Contracting Gastrocnemius Muscle of Goto-Kakizaki Diabetic Rats In Vivo

    PubMed Central

    Macia, Michael; Pecchi, Emilie; Vilmen, Christophe; Desrois, Martine; Lan, Carole; Portha, Bernard; Bernard, Monique; Bendahan, David; Giannesini, Benoît

    2015-01-01

    Insulin resistance, altered lipid metabolism and mitochondrial dysfunction in skeletal muscle would play a major role in type 2 diabetes mellitus (T2DM) development, but the causal relationships between these events remain conflicting. To clarify this issue, gastrocnemius muscle function and energetics were investigated throughout a multidisciplinary approach combining in vivo and in vitro measurements in Goto-Kakizaki (GK) rats, a non-obese T2DM model developing peripheral insulin resistant without abnormal level of plasma non-esterified fatty acids (NEFA). Wistar rats were used as controls. Mechanical performance and energy metabolism were assessed strictly non-invasively using magnetic resonance (MR) imaging and 31-phosphorus MR spectroscopy (31P-MRS). Compared with control group, plasma insulin and glucose were respectively lower and higher in GK rats, but plasma NEFA level was normal. In resting GK muscle, phosphocreatine content was reduced whereas glucose content and intracellular pH were both higher. However, there were not differences between both groups for basal oxidative ATP synthesis rate, citrate synthase activity, and intramyocellular contents for lipids, glycogen, ATP and ADP (an important in vivo mitochondrial regulator). During a standardized fatiguing protocol (6 min of maximal repeated isometric contractions electrically induced at a frequency of 1.7 Hz), mechanical performance and glycolytic ATP production rate were reduced in diabetic animals whereas oxidative ATP production rate, maximal mitochondrial capacity and ATP cost of contraction were not changed. These findings provide in vivo evidence that insulin resistance is not caused by an impairment of mitochondrial function in this diabetic model. PMID:26057538

  12. THEMATICS analysis for functional ion channels

    NASA Astrophysics Data System (ADS)

    Shehadi, Ihsan A.

    Ion channels, as a group of integral membrane proteins, span the cell membrane forming ion-conducting pores that allow ions to traverse the hydrophobic lipid environment rapidly and selectively. The structure of the Streptomyces lividians (KcsA) and Mycobacterium tuberculosis ion channel (Mscl) potassium ion channel have provided the impetus and has helped further the understanding of the structural and functional studies of these channels. The KcsA adapts the voltage-gated mechanism for opening and closing of the channel. While Mcsl represents the mechanosensitive model of the channels. However, the mechanism of the opening and closing of these channels are not fully understood. Electrostatic methods (THEMATICS) are used to locate the site where closing and opening of the channels are controlled. Two clusters of amino acid residues are identified in each of the previously mentioned active models where net charges play an important role in controlling the mechanism of the opening and closure of the ion channels.0

  13. Function analysis for waste information systems

    SciTech Connect

    Sexton, J.L.; Neal, C.T.; Heath, T.C.; Starling, C.D.

    1996-04-01

    This study has a two-fold purpose. It seeks to identify the functional requirements of a waste tracking information system and to find feasible alternatives for meeting those requirements on the Oak Ridge Reservation (ORR) and the Portsmouth (PORTS) and Paducah (PGDP) facilities; identify options that offer potential cost savings to the US government and also show opportunities for improved efficiency and effectiveness in managing waste information; and, finally, to recommend a practical course of action that can be immediately initiated. In addition to identifying relevant requirements, it also identifies any existing requirements that are currently not being completely met. Another aim of this study is to carry out preliminary benchmarking by contacting representative companies about their strategic directions in waste information. The information obtained from representatives of these organizations is contained in an appendix to the document; a full benchmarking effort, however, is beyond the intended scope of this study.

  14. In Vivo Chemical and Structural Analysis of Plant Cuticular Waxes Using Stimulated Raman Scattering Microscopy1[OPEN

    PubMed Central

    Mansfield, Jessica C.; Perfect, Sarah; Seymour, Mark; Smirnoff, Nicholas; Love, John; Moger, Julian

    2015-01-01

    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. PMID:25783412

  15. Endovascular image-guided treatment of in-vivo model aneurysms with asymmetric vascular stents (AVS): evaluation with time-density curve angiographic analysis and histology

    NASA Astrophysics Data System (ADS)

    Dohatcu, A.; Ionita, C. N.; Paciorek, A.; Bednarek, D. R.; Hoffmann, K. R.; Rudin, S.

    2008-03-01

    In this study, we compare the results obtained from Time-Density Curve (TDC) analysis of angiographic imaging sequences with histological evaluation for a rabbit aneurysm model treated with standard stents and new asymmetric vascular stents (AVS) placed by image-guided endovascular deployment. AVSs are stents having a low-porosity patch region designed to cover the aneurysm neck and occlude blood flow inside. To evaluate the AVSs, rabbits with elastase-induced aneurysm models (n=20) were divided into three groups: the first (n=10) was treated with an AVS, the second (n=5) with a non-patch standard coronary stent, and third was untreated as a control (n=5). We used TDC analysis to measure how much contrast media entered the aneurysm before and after treatment. TDCs track contrast-media-density changes as a function of time over the region of interest in x-ray DSA cine-sequences. After 28 days, the animals were sacrificed and the explanted specimens were histologically evaluated. The first group showed an average reduction of contrast flow into the aneurysm of 95% after treatment with an AVS with fully developed thrombus at 28 days follow-up. The rabbits treated with standard stents showed an increase in TDC residency time after treatment and partial-thrombogenesis. The untreated control aneurysms displayed no reduction in flow and were still patent at follow-up. The quantitative TDC analysis findings were confirmed by histological evaluation suggesting that the new AVS has great potential as a definitive treatment for cerebro-vascular aneurysms and that angiographic TDC analysis can provide in-vivo verification.

  16. Multi-resolution analysis generated by a seed function

    E-print Network

    Fabio Bagarello

    2009-04-17

    In this paper we use the equivalence result originally proved by the author which relates a multi-resolution analysis (MRA) of $L^2(R)$ and an orthonormal set of single electron wave-functions in the lowest Landau level, to build up a procedure which produces, starting with a certain square-integrable function, a MRA of $L^2(R)$

  17. Analysis of Multiple Manding Topographies during Functional Communication Training

    ERIC Educational Resources Information Center

    Harding, Jay W.; Wacker, David P.; Berg, Wendy K.; Winborn-Kemmerer, Lisa; Lee, John F.; Ibrahimovic, Muska

    2009-01-01

    We evaluated the effects of reinforcing multiple manding topographies during functional communication training (FCT) to decrease problem behavior for three preschool-age children. During Phase 1, a functional analysis identified conditions that maintained problem behavior for each child. During Phase 2, the children's parents taught them to…

  18. Functional Analysis and Treatment of Inappropriate Sexual Behavior.

    ERIC Educational Resources Information Center

    Fyffe, Christie E.; Kahng, SungWoo; Fittro, Ellen; Russell, David

    2004-01-01

    The results of a functional analysis showed that inappropriate sexual behaviors exhibited by a 9-year-old boy who had been diagnosed with traumatic brain injury were maintained by positive reinforcement in the form of social attention. An intervention consisting of functional communication training and extinction resulted in reduced levels of…

  19. A Top Level Analysis of Training Management Functions.

    ERIC Educational Resources Information Center

    Ackerson, Jack

    1995-01-01

    Presents an approach to conducting a top-level analysis of training management functions when there are problems within a training system. Highlights include data gathering and analyses of a training system; training management functions and activities, including planning, staffing, development, and evaluation and feedback; and development of root…

  20. A Top Level Analysis of Training Management Functions.

    ERIC Educational Resources Information Center

    Ackerson, Jack

    1995-01-01

    Discusses how to conduct a top-level analysis of training management functions to identify problems within a training system resulting from rapid growth, the acquisition of new departments, or mergers. The data gathering process and analyses are explained, training management functions and activities are described, and root causes and solutions…

  1. The Analysis of a#nely Equivalent Boolean Functions #

    E-print Network

    International Association for Cryptologic Research (IACR)

    for the following two reasons: first, equivalent functions have similar properties (like Hamming weight distributionThe Analysis of a#nely Equivalent Boolean Functions # Qing­shu Meng Min Yang Huan­guo Zhang Yuzhen#nely equivalent and to obtain the a#ne equivalence relationship if they are equivalent. For example, all 8

  2. The Effects of Reinforcement Magnitude on Functional Analysis Outcomes

    ERIC Educational Resources Information Center

    Volkert, Valerie M.; Lerman, Dorothea C.; Vorndran, Christina

    2005-01-01

    The duration or magnitude of reinforcement has varied and often appears to have been selected arbitrarily in functional analysis research. Few studies have evaluated the effects of reinforcement magnitude on problem behavior, even though basic findings indicate that this parameter may affect response rates during functional analyses. In the…

  3. Fractal analysis of resting state functional connectivity of the brain

    E-print Network

    Fractal analysis of resting state functional connectivity of the brain Wonsang You1 , Sophie Achard neuroimaging data tend to exhibit fractal behavior where their power spectrums follow power-law scaling. Resting state functional connectivity is signicantly inuenced by fractal behav- ior which may not directly

  4. Quantitative in vivo analysis of small bowel motility using MRI examinations in mice--proof of concept study.

    PubMed

    Bickelhaupt, S; Wurnig, M C; Lesurtel, M; Patak, M A; Boss, A

    2015-01-01

    Small bowel motility analyses using magnetic resonance imaging (MRI) could reduce current invasive techniques in animal studies and comply with the 'three Rs' rule for human animal experimentation. Thus we investigated the feasibility of in vivo small bowel motility analyses in mice using dynamic MRI acquisitions. All experimental procedures were approved by the institutional animal care committee. Six C57BL/6 mice underwent MRI without additional preparation after isoflurane anaesthetization in the prone position on a 4.7 T small animal imager equipped with a linear polarized hydrogen birdcage whole-body mouse coil. Motility was assessed using a true fast imaging in a steady precession sequence in the coronal orientation (acquisition time per slice 512?ms, in-plane resolution 234?×?234?µm, matrix size 128?×?128, slice thickness 1?mm) over 30?s corresponding to 60 acquisitions. Motility was manually assessed measuring the small bowel diameter change over time. The resulting motility curves were analysed for the following parameters: contraction frequency per minute (cpm), maximal contraction amplitude (maximum to minimum [mm]), luminal diameter (mm) and luminal occlusion rate. Small bowel motility quantification was found to be possible in all animals with a mean small bowel contraction frequency of 10.67?cpm (SD?±?3.84), a mean amplitude of the contractions of 1.33?mm (SD?±?0.43) and a mean luminal diameter of 1.37?mm (SD?±?0.42). The mean luminal occlusion rate was 1.044 (SD?±?0.45%/100). The mean duration needed for a single motility assessment was 185?s (SD?±?54.02). Thus our study demonstrated the feasibility of an easy and time-sparing functional assessment for in vivo small bowel motility analyses in mice. This could improve the development of small animal models of intestinal diseases and provide a method similar to clinical MR examinations that is in concordance with the 'three Rs' for humane animal experimentation. PMID:25266965

  5. Ex Vivo Chemical Cytometric Analysis of Protein Tyrosine Phosphatase Activity in Single Human Airway Epithelial Cells

    PubMed Central

    Phillips, Ryan M.; Dailey, Lisa A.; Bair, Eric; Samet, James M.; Allbritton, Nancy L.

    2014-01-01

    We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min?1 mg?1) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn2+, and 1,2-naphthoquinone (76%, 69%, 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min?1 mg?1, and was also effectively inhibited by pre-incubation of the cells with the inhibitors pervanadate, Zn2+, and 1,2- naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min?1 mg?1 resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34–36 pmol min?1 mg?1 (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues. PMID:24380370

  6. Regulation of prostaglandin production by nitric oxide; an in vivo analysis.

    PubMed Central

    Salvemini, D; Settle, S L; Masferrer, J L; Seibert, K; Currie, M G; Needleman, P

    1995-01-01

    1. Endotoxin E. Coli lipopolysaccharide (LPS)-treatment in conscious, restrained rats increased plasma and urinary prostaglandin (PG) and nitric oxide (NO) production. Inducible cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS) expression accounted for the LPS-induced PG and NO release since the glucocorticoid, dexamethasone inhibited both effects. Thus, LPS (4 mg kg-1) increased the plasma levels of nitrite/nitrate from 14 +/- 1 to 84 +/- 7 microM within 3 h and this rise was inhibited to 35 +/- 1 microM by dexamethasone. Levels of 6-keto PGF1 alpha in the plasma were below the detection limit of the assay (< 0.2 ng ml-1). However, 3 h after the injection of LPS these levels rose to 2.6 +/- 0.2 ng ml-1 and to 0.7 +/- 0.01 ng ml-1 after LPS in rats that received dexamethasone. 2. The induced enzymes were inhibited in vivo with selective COX and NOS inhibitors. Furthermore, NOS inhibitors, that did not affect COX activity in vitro markedly suppressed PG production in the LPS-treated animals. For instance, the LPS-induced increased in plasma nitrite/nitrate and 6-keto PGF1 alpha at 3 h was decreased to 18 +/- 2 microM and 0.5 +/- 0.02 ng ml-1, 23 +/- 1 microM and 0.7 +/- 0.01 ng ml-1, 29 +/- 2 microM and 1 +/- 0.01 ng ml-1 in rats treated with LPS in the presence of the NOS inhibitors NG-monomethyl-L-arginine, NG-nitro arginine methyl ester and aminoguanidine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7542531

  7. RESEARCH ARTICLE Open Access Development and analysis of an in vivo-

    E-print Network

    , as the analysis suggested, lipids are used as the main source for carbon metabolism and oxygen must be available to life in an acidic environment, highlight the importance of different enzymes in the tricarboxylic acid-cycle

  8. GENETIC ANALYSYS OF THE IN VIVO FUNCTION AND REGULATION OF THE ONCOPROTEIN CDK8 IN DROSOPHILA 

    E-print Network

    Bridges, Lauren

    2012-05-09

    . Importantly, both CDK8 and CycC are highly conserved in all eukaryotes. Thus we use Drosophila as an experimental system to identify both the upstream regulators and downstream effectors of CDK8. We expect to elucidate the fundamental roles of CDK8- Cyc...

  9. GENETIC ANALYSYS OF THE IN VIVO FUNCTION AND REGULATION OF THE ONCOPROTEIN CDK8 IN DROSOPHILA 

    E-print Network

    Park, Gee Yoon

    2012-05-09

    Cyclin-Dependent Kinase 8 (CDK8) has recently emerged as an oncoprotein that is amplified in both colorectal and melanoma cancers. The importance of CDK8 and its regulatory partner Cyclin C (CycC) is highlighted by the clinical observations showing...

  10. Whole-genome sequencing and microarray analysis of ex vivo Plasmodium vivax reveal selective pressure on putative drug resistance genes.

    PubMed

    Dharia, Neekesh V; Bright, A Taylor; Westenberger, Scott J; Barnes, S Whitney; Batalov, Serge; Kuhen, Kelli; Borboa, Rachel; Federe, Glenn C; McClean, Colleen M; Vinetz, Joseph M; Neyra, Victor; Llanos-Cuentas, Alejandro; Barnwell, John W; Walker, John R; Winzeler, Elizabeth A

    2010-11-16

    Plasmodium vivax causes 25-40% of malaria cases worldwide, yet research on this human malaria parasite has been neglected. Nevertheless, the recent publication of the P. vivax reference genome now allows genomics and systems biology approaches to be applied to this pathogen. We show here that whole-genome analysis of the parasite can be achieved directly from ex vivo-isolated parasites, without the need for in vitro propagation. A single isolate of P. vivax obtained from a febrile patient with clinical malaria from Peru was subjected to whole-genome sequencing (30× coverage). This analysis revealed over 18,261 single-nucleotide polymorphisms (SNPs), 6,257 of which were further validated using a tiling microarray. Within core chromosomal genes we find that one SNP per every 985 bases of coding sequence distinguishes this recent Peruvian isolate, designated IQ07, from the reference Salvador I strain obtained in 1972. This full-genome sequence of an uncultured P. vivax isolate shows that the same regions with low numbers of aligned sequencing reads are also highly variable by genomic microarray analysis. Finally, we show that the genes containing the largest ratio of nonsynonymous-to-synonymous SNPs include two AP2 transcription factors and the P. vivax multidrug resistance-associated protein (PvMRP1), an ABC transporter shown to be associated with quinoline and antifolate tolerance in Plasmodium falciparum. This analysis provides a data set for comparative analysis with important potential for identifying markers for global parasite diversity and drug resistance mapping studies. PMID:21037109

  11. In vivo dose response and in vitro mechanistic analysis of enhanced immunoglobulin A production by Lactobacillus plantarum AYA

    PubMed Central

    KIKUCHI, Yosuke; YOSHIDA, Hikaru; OGITA, Tasuku; OKITA, Kimiko; FUKUDOME, Shin-ichi; SUZUKI, Takuya; TANABE, Soichi

    2015-01-01

    Secretory immunoglobulin A (IgA) mediates the mucosal immune system, which provides the first line of defense against inhaled and ingested pathogenic bacteria and viruses. Lactobacillus plantarum AYA increases the IgA level of Peyer’s patch (PP) cells, but the recommended amount of consumption and the mechanism of action remains unclear. Better understanding of these is essential to development of L. plantarum AYA for use in functional foods. Therefore, we investigated the dose-response effect (in vivo) and mechanism (in vitro) of IgA enhancement induced by L. plantarum AYA. In the small intestine of the mice fed a diet containing 0.03% or 0.3% of L. plantarum AYA powder for 4 weeks, the IgA levels were significantly increased. Thus, it is suggested that the recommended amount of consumption of L. plantarum AYA is about 0.72?mg per day. In addition, the bacterial cell wall fraction significantly enhanced the IgA production level of murine PP cells in the in vitro assay. The ability of whole cells and the cell wall fraction to enhance IgA levels was significantly inhibited by an anti-Toll-like receptor-2 (TLR-2) antibody, which suggests that the cell wall fraction of L. plantarum AYA increases the IgA level via TLR-2. These findings indicate that L. plantarum AYA is a potential functional food source that maintains mucosal immunity. PMID:26221576

  12. In vivo dose response and in vitro mechanistic analysis of enhanced immunoglobulin A production by Lactobacillus plantarum AYA.

    PubMed

    Kikuchi, Yosuke; Yoshida, Hikaru; Ogita, Tasuku; Okita, Kimiko; Fukudome, Shin-Ichi; Suzuki, Takuya; Tanabe, Soichi

    2015-01-01

    Secretory immunoglobulin A (IgA) mediates the mucosal immune system, which provides the first line of defense against inhaled and ingested pathogenic bacteria and viruses. Lactobacillus plantarum AYA increases the IgA level of Peyer's patch (PP) cells, but the recommended amount of consumption and the mechanism of action remains unclear. Better understanding of these is essential to development of L. plantarum AYA for use in functional foods. Therefore, we investigated the dose-response effect (in vivo) and mechanism (in vitro) of IgA enhancement induced by L. plantarum AYA. In the small intestine of the mice fed a diet containing 0.03% or 0.3% of L. plantarum AYA powder for 4 weeks, the IgA levels were significantly increased. Thus, it is suggested that the recommended amount of consumption of L. plantarum AYA is about 0.72?mg per day. In addition, the bacterial cell wall fraction significantly enhanced the IgA production level of murine PP cells in the in vitro assay. The ability of whole cells and the cell wall fraction to enhance IgA levels was significantly inhibited by an anti-Toll-like receptor-2 (TLR-2) antibody, which suggests that the cell wall fraction of L. plantarum AYA increases the IgA level via TLR-2. These findings indicate that L. plantarum AYA is a potential functional food source that maintains mucosal immunity. PMID:26221576

  13. Phosphorylation of Enabled by the Drosophila Abelson Tyrosine Kinase Regulates the In Vivo Function and Protein-Protein Interactions of Enabled

    PubMed Central

    Comer, Allen R.; Ahern-Djamali, Shawn M.; Juang, Jyh-Lyh; Jackson, P. David; Hoffmann, F. M.

    1998-01-01

    Drosophila Enabled (Ena) is a member of a family of cytoskeleton-associated proteins including mammalian vasodilator-stimulated phosphoprotein and murine Enabled that regulate actin cytoskeleton assembly. Mutations in Drosophila ena were discovered as dominant genetic suppressors of mutations in the Abelson tyrosine kinase (Abl), suggesting that Ena and Abl function in the same pathway or process. We have identified six tyrosine residues on Ena that are phosphorylated by Abl in vitro and in vivo. Mutation of these phosphorylation sites to phenylalanine partially impaired the ability of Ena to restore viability to ena mutant animals, indicating that phosphorylation is required for optimal Ena function. Phosphorylation of Ena by Abl inhibited the binding of Ena to SH3 domains in vitro, suggesting that one effect of Ena phosphorylation may be to modulate its association with other proteins. PMID:9418863

  14. RNA-Seq analysis uncovers transcriptomic variations between morphologically similar in vivo- and in vitro-derived bovine blastocysts

    PubMed Central

    2012-01-01

    Background A valuable tool for both research and industry, in vitro fertilization (IVF) has applications range from gamete selection and preservation of traits to cloning. Although IVF has achieved worldwide use, with approximately 339,685 bovine embryos transferred in 2010 alone, there are still continuing difficulties with efficiency. It is rare to have more than 40% of fertilized in vitro cattle oocytes reach blastocyst stage by day 8 of culture, and pregnancy rates are reported as less than 45% for in vitro produced embryos. To investigate potential influences in-vitro fertilization (IVF) has on embryonic development, this study compares in vivo- and in vitro-derived bovine blastocysts at a similar stage and quality grade (expanded, excellent quality) to determine the degree of transcriptomic variation beyond morphology using RNA-Seq. Results A total of 26,906,451 and 38,184,547 fragments were sequenced for in vitro and in vivo embryo pools, respectively. We detected expression for a total of 17,634 genes, with 793 genes showing differential expression between the two embryo populations with false discovery rate (FDR) < 0.05. There were also 395 novel transcribed units found, of which 45 were differentially expressed (FDR < 0.05). In addition, 4,800 genes showed evidence of alternative splicing, with 873 genes displaying differential alternative splicing between the two pools (FDR < 0.05). Using GO enrichment analysis, multiple biological pathways were found to be significantly enriched for differentially expressed genes (FDR < 0.01), including cholesterol and sterol synthesis, system development, and cell differentiation. Conclusions Thus, our results support that IVF may influence at the transcriptomic level and that morphology is limited in full characterization of bovine preimplantation embryos. PMID:22452724

  15. Tabebuia avellanedae naphthoquinones: activity against methicillin-resistant staphylococcal strains, cytotoxic activity and in vivo dermal irritability analysis

    PubMed Central

    Pereira, Eliezer Menezes; Machado, Thelma de Barros; Leal, Ivana Correa Ramos; Jesus, Desyreé Murta; Damaso, Clarissa Rosa de Almeida; Pinto, Antonio Ventura; Giambiagi-deMarval, Marcia; Kuster, Ricardo Machado; dos Santos, Kátia Regina Netto

    2006-01-01

    Background Methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococcus infections are a worldwide concern. Currently, these isolates have also shown resistance to vancomycin, the last therapy used in these cases. It has been observed that quinones and other related compounds exhibit antibacterial activity. This study evaluated the antibacterial activity, toxicity and in vivo dermal irritability of lapachol extracted from Tabebuia avellanedae and derivatives against methicillin-resistant staphylococcal isolates. In addition, its mechanism of action was also analyzed. Methods The compounds ?-lapachone, 3-hydroxy ? N lapachone and ?-lapachone were tested to determine the MIC values against methicillin-resistant S. aureus, S. epidermidis and S. haemolyticus strains, being the two last ones hetero-resistant to vancomycin. Experiments of protein synthesis analysis to investigate the naphthoquinones action were assessed. In vitro toxicity to eukaryotic BSC-40 African Green Monkey Kidney cell cultures and in vivo primary dermal irritability in healthy rabbits were also performed. Results The compounds tested showed antibacterial activity (MICs of 8, 4/8 and 64/128 ?g/mL to ?-lapachone, 3-hydroxy ? N lapachone and ?-lapachone, respectively), but no bactericidal activity was observed (MBC > 512 ?g/mL for all compounds). Although it has been observed toxic effect in eukaryotic cells, the compounds were shown to be atoxic when applied as topic preparations in healthy rabbits. No inhibition of proteins synthesis was observed. Conclusion Our results suggest that quinones could be used in topic preparations against wound infections caused by staphylococci, after major investigation of the pharmacological properties of the compounds. Studies about the use of these compounds on tumoral cells could be carried on, due to their effect in eukaryotic cells metabolism. PMID:16553949

  16. Fourier functional analysis for unsteady aerodynamic modeling

    NASA Technical Reports Server (NTRS)

    Lan, C. Edward; Chin, Suei

    1991-01-01

    A method based on Fourier analysis is developed to analyze the force and moment data obtained in large amplitude forced oscillation tests at high angles of attack. The aerodynamic models for normal force, lift, drag, and pitching moment coefficients are built up from a set of aerodynamic responses to harmonic motions at different frequencies. Based on the aerodynamic models of harmonic data, the indicial responses are formed. The final expressions for the models involve time integrals of the indicial type advocated by Tobak and Schiff. Results from linear two- and three-dimensional unsteady aerodynamic theories as well as test data for a 70-degree delta wing are used to verify the models. It is shown that the present modeling method is accurate in producing the aerodynamic responses to harmonic motions and the ramp type motions. The model also produces correct trend for a 70-degree delta wing in harmonic motion with different mean angles-of-attack. However, the current model cannot be used to extrapolate data to higher angles-of-attack than that of the harmonic motions which form the aerodynamic model. For linear ramp motions, a special method is used to calculate the corresponding frequency and phase angle at a given time. The calculated results from modeling show a higher lift peak for linear ramp motion than for harmonic ramp motion. The current model also shows reasonably good results for the lift responses at different angles of attack.

  17. Time-frequency analysis and Harmonic Gaussian Functions

    E-print Network

    Tokiniaina Ranaivoson; Raoelina Andriambololona; Rakotoson Hanitriarivo

    2013-08-08

    A method for time-frequency analysis is given. The approach utilizes properties of Gaussian distribution, properties of Hermite polynomials and Fourier analysis. We begin by the definitions of a set of functions called harmonic Gaussian functions. Then these functions are used to define a set of transformations,noted T_n, which associate to a function {\\psi},of the time variable t, a set of functions {\\Psi}_n which depend on time, frequency and frequency (or time) standard deviation. Some properties of the transformations T_n and the functions {\\Psi}_n are given. It is proved in particular that the square of the modulus of each function {\\Psi}_n can be interpreted as a representation of the energy distribution of the signal, represented by the function {\\psi}, in the time-frequency plane for a given value of the frequency (or time) standard deviation. It is also shown that the function {\\psi}, can be recovered from the functions{\\Psi}_n.

  18. Protein Polymer MRI Contrast Agents: Longitudinal Analysis of Biomaterials In Vivo

    E-print Network

    Barron, Annelise E.

    to regenerate biological function by combining cells with material supports, development is hindered include the use of numerous animals, a lack of three- dimensional data, artifacts from the animal and a favorable safety profile. It is an excellent imaging mo- dality for longitudinal noninvasive assessment

  19. Graph theoretical analysis of structural and functional connectivity MRI in normal and pathological brain networks.

    PubMed

    Guye, Maxime; Bettus, Gaelle; Bartolomei, Fabrice; Cozzone, Patrick J

    2010-12-01

    Graph theoretical analysis of structural and functional connectivity MRI data (ie. diffusion tractography or cortical volume correlation and resting-state or task-related (effective) fMRI, respectively) has provided new measures of human brain organization in vivo. The most striking discovery is that the whole-brain network exhibits "small-world" properties shared with many other complex systems (social, technological, information, biological). This topology allows a high efficiency at different spatial and temporal scale with a very low wiring and energy cost. Its modular organization also allows for a high level of adaptation. In addition, degree distribution of brain networks demonstrates highly connected hubs that are crucial for the whole-network functioning. Many of these hubs have been identified in regions previously defined as belonging to the default-mode network (potentially explaining the high basal metabolism of this network) and the attentional networks. This could explain the crucial role of these hub regions in physiology (task-related fMRI data) as well as in pathophysiology. Indeed, such topological definition provides a reliable framework for predicting behavioral consequences of focal or multifocal lesions such as stroke, tumors or multiple sclerosis. It also brings new insights into a better understanding of pathophysiology of many neurological or psychiatric diseases affecting specific local or global brain networks such as epilepsy, Alzheimer's disease or schizophrenia. Graph theoretical analysis of connectivity MRI data provides an outstanding framework to merge anatomical and functional data in order to better understand brain pathologies. PMID:20349109

  20. Anti-atherosclerotic function of Astragali Radix extract: downregulation of adhesion molecules in vitro and in vivo

    PubMed Central

    2012-01-01

    Background Atherosclerosis is considered to be a chronic inflammatory disease. Astragali Radix extract (ARE) is one of the major active ingredients extracted from the root of Astragalus membranaceus Bge. Although ARE has an anti-inflammatory function, its anti-atherosclerotic effects and mechanisms have not yet been elucidated. Methods Murine endothelial SVEC4-10 cells were pretreated with different doses of ARE at different times prior to induction with tumor necrosis factor (TNF)-?. Cell adhesion assays were performed using THP-1 cells and assessed by enzyme-linked immunosorbent assay, western blotting and immunofluorescence analyses to detect the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), phosphorylated inhibitor of ?B (p-i?B) and nuclear factor (NF)-?B. We also examined the effect of ARE on atherosclerosis in the aortic endothelium of apolipoprotein E-deficient (apoE?/?) mice. Results TNF-? strongly increased the expression of VCAM-1 and ICAM-1 accompanied by increased expression of p-i?B and NF-?B proteins. However, the expression levels of VCAM-1 and ICAM-1 were reduced by ARE in dose- and time-dependent manners, with the strongest effect at a dose of 120??g/ml incubated for 4?h. This was accompanied by significantly decreased expression of p-i?B and inhibited activation of NF-?B. Immunofluorescence analysis also revealed that oral administration of ARE resulted in downregulation of adhesion molecules and decreased expression of macrophages in the aortic endothelium of apoE?/? mice. ARE could suppress the inflammatory reaction and inhibit the progression of atherosclerotic lesions in apoE?/? mice. Conclusion This study demonstrated that ARE might be an effective anti-inflammatory agent for the treatment of atherosclerosis, possibly acting via the decreased expression of adhesion molecules. PMID:22536886

  1. Integrating structural and functional imaging for computer assisted detection of prostate cancer on multi-protocol in vivo 3 Tesla MRI

    NASA Astrophysics Data System (ADS)

    Viswanath, Satish; Bloch, B. Nicolas; Rosen, Mark; Chappelow, Jonathan; Toth, Robert; Rofsky, Neil; Lenkinski, Robert; Genega, Elizabeth; Kalyanpur, Arjun; Madabhushi, Anant

    2009-02-01

    Screening and detection of prostate cancer (CaP) currently lacks an image-based protocol which is reflected in the high false negative rates currently associated with blinded sextant biopsies. Multi-protocol magnetic resonance imaging (MRI) offers high resolution functional and structural data about internal body structures (such as the prostate). In this paper we present a novel comprehensive computer-aided scheme for CaP detection from high resolution in vivo multi-protocol MRI by integrating functional and structural information obtained via dynamic-contrast enhanced (DCE) and T2-weighted (T2-w) MRI, respectively. Our scheme is fully-automated and comprises (a) prostate segmentation, (b) multimodal image registration, and (c) data representation and multi-classifier modules for information fusion. Following prostate boundary segmentation via an improved active shape model, the DCE/T2-w protocols and the T2-w/ex vivo histological prostatectomy specimens are brought into alignment via a deformable, multi-attribute registration scheme. T2-w/histology alignment allows for the mapping of true CaP extent onto the in vivo MRI, which is used for training and evaluation of a multi-protocol MRI CaP classifier. The meta-classifier used is a random forest constructed by bagging multiple decision tree classifiers, each trained individually on T2-w structural, textural and DCE functional attributes. 3-fold classifier cross validation was performed using a set of 18 images derived from 6 patient datasets on a per-pixel basis. Our results show that the results of CaP detection obtained from integration of T2-w structural textural data and DCE functional data (area under the ROC curve of 0.815) significantly outperforms detection based on either of the individual modalities (0.704 (T2-w) and 0.682 (DCE)). It was also found that a meta-classifier trained directly on integrated T2-w and DCE data (data-level integration) significantly outperformed a decision-level meta-classifier, constructed by combining the classifier outputs from the individual T2-w and DCE channels.

  2. Analysis of cell movements in zebrafish embryos: morphometrics and measuring movement of labeled cell populations in vivo.

    PubMed

    Sepich, Diane S; Solnica-Krezel, Lilianna

    2005-01-01

    Cell movements occur in all phases of animal life from embryogenesis, to maintaining adult organs, to comprising a critical component of pathology. During gastrulation, cells demonstrate a repertoire of morphogenetic movements coordinated with fate inductions to sculpt the embryonic body. The morphogenetic behaviors, underlying mechanisms, and their control, are the subject of much current study. External development of the transparent zebrafish embryo, the abundance of mutations influencing cell movements, as well as a range of observation and manipulation methods, make the zebrafish valuable for cell movement studies. This chapter offers a conceptual background for analysis of gastrulation cell movements by reviewing how region specific cell movements shape the wild-type zebrafish embryo, and how defective morphogenetic movements alone or in combination with altered cell fate specification distort the body plans of known zebrafish mutants. We furnish methods for the morphometric analysis of embryonic shape and organ rudiments in live and fixed embryos, and present data collected from live wild-type, dorsoventral patterning (somitabun and chordino) and convergence and extension (knypek and trilobite) classes of mutants. We provide a method for quantitative assessment of the movements of cell populations in vivo, and a method for determining whether cell fate and/or movement are disturbed. PMID:15576915

  3. Development and validation of technique for in-vivo 3D analysis of cranial bone graft survival

    NASA Astrophysics Data System (ADS)

    Bernstein, Mark P.; Caldwell, Curtis B.; Antonyshyn, Oleh M.; Ma, Karen; Cooper, Perry W.; Ehrlich, Lisa E.

    1997-05-01

    Bone autografts are routinely employed in the reconstruction of facial deformities resulting from trauma, tumor ablation or congenital malformations. The combined use of post- operative 3D CT and SPECT imaging provides a means for quantitative in vivo evaluation of bone graft volume and osteoblastic activity. The specific objectives of this study were: (1) Determine the reliability and accuracy of interactive computer-assisted analysis of bone graft volumes based on 3D CT scans; (2) Determine the error in CT/SPECT multimodality image registration; (3) Determine the error in SPECT/SPECT image registration; and (4) Determine the reliability and accuracy of CT-guided SPECT uptake measurements in cranial bone grafts. Five human cadaver heads served as anthropomorphic models for all experiments. Four cranial defects were created in each specimen with inlay and onlay split skull bone grafts and reconstructed to skull and malar recipient sites. To acquire all images, each specimen was CT scanned and coated with Technetium doped paint. For purposes of validation, skulls were landmarked with 1/16-inch ball-bearings and Indium. This study provides a new technique relating anatomy and physiology for the analysis of cranial bone graft survival.

  4. Prolate Spheroidal Wave Functions In q-Fourier Analysis

    E-print Network

    Lazhar Dhaouadi

    2008-04-09

    The prolate spheroidal wave functions, which are a special case of the spheroidal wave functions, possess a very surprising and unique property [6]. They are an orthogonal basis of both $L^2(-1,1)$ and the Paley-Wiener space of bandlimited functions. They also satisfy a discrete orthogonality relation. No other system of classical orthogonal functions is known to possess this strange property. We prove that there are new systems possessing this property in $q$-Fourier analysis. As application we give a new sampling formula with $q^n$ as sampling points, where 0 < q < 1.

  5. Reproducibility of In Vivo Corneal Confocal Microscopy Using an Automated Analysis Program for Detection of Diabetic Sensorimotor Polyneuropathy

    PubMed Central

    Ostrovski, Ilia; Lovblom, Leif E.; Farooqi, Mohammed A.; Scarr, Daniel; Boulet, Genevieve; Hertz, Paul; Wu, Tong; Halpern, Elise M.; Ngo, Mylan; Ng, Eduardo; Orszag, Andrej; Bril, Vera; Perkins, Bruce A.

    2015-01-01

    Objective In vivo Corneal Confocal Microscopy (IVCCM) is a validated, non-invasive test for diabetic sensorimotor polyneuropathy (DSP) detection, but its utility is limited by the image analysis time and expertise required. We aimed to determine the inter- and intra-observer reproducibility of a novel automated analysis program compared to manual analysis. Methods In a cross-sectional diagnostic study, 20 non-diabetes controls (mean age 41.4±17.3y, HbA1c 5.5±0.4%) and 26 participants with type 1 diabetes (42.8±16.9y, 8.0±1.9%) underwent two separate IVCCM examinations by one observer and a third by an independent observer. Along with nerve density and branch density, corneal nerve fibre length (CNFL) was obtained by manual analysis (CNFLMANUAL), a protocol in which images were manually selected for automated analysis (CNFLSEMI-AUTOMATED), and one in which selection and analysis were performed electronically (CNFLFULLY-AUTOMATED). Reproducibility of each protocol was determined using intraclass correlation coefficients (ICC) and, as a secondary objective, the method of Bland and Altman was used to explore agreement between protocols. Results Mean CNFLManual was 16.7±4.0, 13.9±4.2 mm/mm2 for non-diabetes controls and diabetes participants, while CNFLSemi-Automated was 10.2±3.3, 8.6±3.0 mm/mm2 and CNFLFully-Automated was 12.5±2.8, 10.9 ± 2.9 mm/mm2. Inter-observer ICC and 95% confidence intervals (95%CI) were 0.73(0.56, 0.84), 0.75(0.59, 0.85), and 0.78(0.63, 0.87), respectively (p = NS for all comparisons). Intra-observer ICC and 95%CI were 0.72(0.55, 0.83), 0.74(0.57, 0.85), and 0.84(0.73, 0.91), respectively (p<0.05 for CNFLFully-Automated compared to others). The other IVCCM parameters had substantially lower ICC compared to those for CNFL. CNFLSemi-Automated and CNFLFully-Automated underestimated CNFLManual by mean and 95%CI of 35.1(-4.5, 67.5)% and 21.0(-21.6, 46.1)%, respectively. Conclusions Despite an apparent measurement (underestimation) bias in comparison to the manual strategy of image analysis, fully-automated analysis preserves CNFL reproducibility. Future work must determine the diagnostic thresholds specific to the fully-automated measure of CNFL. PMID:26539984

  6. In vivo labeling and specific magnetic bead separation of RNA for biofilm characterization and stress-induced gene expression analysis in bacteria.

    PubMed

    Stankiewicz, Nikolai; Gold, Andrea; Yüksel, Yousra; Berensmeier, Sonja; Schwartz, Thomas

    2009-12-01

    The method of in vivo labeling and separation of bacterial RNA was developed as an approach to elucidating the stress response of natural bacterial populations. This technique is based on the incorporation of digoxigenin-11-uridine-5'-triphosphate (DIG-11-UTP) in the RNA of active bacteria. The digoxigenin fulfills a dual role as a label of de novo synthesized RNA and a target for magnetic bead separation from a total RNA extract. Depending on the growth conditions and the population's composition, the assembly rate of DIG-11-UTP ranged from 1.2% to 12.5% of the total RNA in gram-positive and gram-negative reference bacteria as well as in natural biofilms from drinking water, surface water, and lake sediment. Separation of DIG-RNA from total RNA extracts was performed with a biotinylated anti-digoxigenin antibody and streptavidin-functionalized magnetic particles. The average separation yield from total RNA extracts was about 95% of labeled RNA. The unspecific bindings of non-labeled nucleic acids were smaller than 0.2%, as was evaluated by spiking experiments with an unmarked DNA amplicon. Applicability of the method developed was demonstrated by rRNA-directed PCR-DGGE population analysis of natural biofilms and expression profiling of two stress-induced genes (vanA and rpoS) in reference bacteria. PMID:19837116

  7. In Vitro and In Vivo Analysis of the Gram-Negative Bacteria-Derived Riboflavin Precursor Derivatives Activating Mouse MAIT Cells.

    PubMed

    Soudais, Claire; Samassa, Fatoumata; Sarkis, Manal; Le Bourhis, Lionel; Bessoles, Stéphanie; Blanot, Didier; Hervé, Mireille; Schmidt, Frédéric; Mengin-Lecreulx, Dominique; Lantz, Olivier

    2015-05-15

    Mucosal-associated invariant T (MAIT) cells recognize microbial compounds presented by the MHC-related 1 (MR1) protein. Although riboflavin precursor derivatives from Gram-positive bacteria have been characterized, some level of ligand heterogeneity has been suggested through the analysis of the MAIT cell TCR repertoire in humans and differential reactivity of human MAIT cell clones according to the bacteria. In this study, using Gram-negative bacteria mutated for the riboflavin biosynthetic pathway, we show a strict correlation between the ability to synthesize the 5-amino-ribityl-uracil riboflavin precursor and to activate polyclonal and quasi-monoclonal mouse MAIT cells. To our knowledge, we show for the first time that the semipurified bacterial fraction and the synthetic ligand activate murine MAIT cells in vitro and in vivo. We describe new MR1 ligands that do not activate MAIT cells but compete with bacterial and synthetic compounds activating MAIT cells, providing the capacity to modulate MAIT cell activation. Through competition experiments, we show that the most active synthetic MAIT cell ligand displays the same functional avidity for MR1 as does the microbial compound. Altogether, these results show that most, if not all, MAIT cell ligands found in Escherichia coli are related to the riboflavin biosynthetic pathway and display very limited heterogeneity. PMID:25870247

  8. In Vitro Differentiation of Pluripotent Stem Cells into Functional ? Islets Under 2D and 3D Culture Conditions and In Vivo Preclinical Validation of 3D Islets.

    PubMed

    Bose, Bipasha; Sudheer, P Shenoy

    2016-01-01

    Since the advent of pluripotent stem cells, (embryonic and induced pluripotent stem cells), applications of such pluripotent stem cells are of prime importance. Indeed, scientists are involved in studying the basic biology of pluripotent stem cells, but equal impetus is there to direct the pluripotent stem cells into multiple lineages for cell therapy applications. Scientists across the globe have been successful, to a certain extent, in obtaining cells of definitive endoderm and also pancreatic ? islets by differentiating human pluripotent stem cells. Pluripotent stem cell differentiation protocols aim at mimicking in vivo embryonic development. As in vivo embryonic development is a complex process and involves interplay of multiple cytokines, the differentiation protocols also involve a stepwise use of multiple cytokines. Indeed the novel markers for pancreas organogenesis serve as the roadmaps to develop new protocols for pancreatic differentiation from pluripotent stem cells. Earliest developed protocols for pancreas differentiation involved "Nestin selection pathway," a pathway common for both neuronal and pancreatic differentiation lead to the generation of cells that were a combination of cells from neuronal lineage. Eventually with the discovery of hierarchy of ? cell transcription factors like Pdx1, Pax4, and Nkx2.2, forced expression of such transcription factors proved successful in converting a pluripotent stem cell into a ? cell. Protocols developed almost half a decade ago to the recent ones rather involve stepwise differentiations involving various cytokines and could generate as high as 25 % functional insulin-positive cells in vitro. Most advanced protocols for ? islet differentiations from human pluripotent stem cells focused on 3D culture conditions, which reportedly produced 60-65 % functional ? islet cells. Here, we describe the protocol for differentiation of human pluripotent stem cells into functional ? cells under both 2D and 3D culture conditions. PMID:25783769

  9. Accelerated apoptotic death and in vivo turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2

    PubMed Central

    Lang, Elisabeth; Bissinger, Rosi; Fajol, Abul; Salker, Madhuri S.; Singh, Yogesh; Zelenak, Christine; Ghashghaeinia, Mehrdad; Gu, Shuchen; Jilani, Kashif; Lupescu, Adrian; Reyskens, Kathleen M. S. E.; Ackermann, Teresa F.; Föller, Michael; Schleicher, Erwin; Sheffield, William P.; Arthur, J. Simon C.; Lang, Florian; Qadri, Syed M.

    2015-01-01

    The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk?/?) and corresponding wild-type mice (msk+/+). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk?/? and msk+/+ mice, but reticulocyte count was significantly increased in msk?/? mice. Cell membrane PS exposure was similar in untreated msk?/? and msk+/+ erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk?/? erythrocytes than in msk+/+ erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk?/? erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk?/? mice. The spleens from msk?/? mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk+/+ mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice. PMID:26611568

  10. Investigation of Functional Activity of Cells in Granulomatous Inflammatory Lesions from Mice with Latent Tuberculous Infection in the New Ex Vivo Model

    PubMed Central

    2013-01-01

    The new ex vivo model system measuring functional input of individual granuloma cells to formation of granulomatous inflammatory lesions in mice with latent tuberculous infection has been developed and described in the current study. Monolayer cultures of cells that migrated from individual granulomas were established in the proposed culture settings for mouse spleen and lung granulomas induced by in vivo exposure to BCG vaccine. The cellular composition of individual granulomas was analyzed. The expression of the leukocyte surface markers such as phagocytic receptors CD11b, CD11c, CD14, and CD16/CD32 and the expression of the costimulatory molecules CD80, CD83, and CD86 were tested as well as the production of proinflammatory cytokines (IFN? and IL-1?) and growth factors (GM-CSF and FGFb) for cells of individual granulomas. The colocalization of the phagocytic receptors and costimulatory molecules in the surface microdomains of granuloma cells (with and without acid-fast BCG-mycobacteria) has also been detected. It was found that some part of cytokine macrophage producers have carried acid-fast mycobacteria. Detected modulation in dynamics of production of pro-inflammatory cytokines, growth factors, and leukocyte surface markers by granuloma cells has indicated continued processes of activation and deactivation of granuloma inflammation cells during the latent tuberculous infection progress in mice. PMID:24198843

  11. Accelerated apoptotic death and in vivo turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2.

    PubMed

    Lang, Elisabeth; Bissinger, Rosi; Fajol, Abul; Salker, Madhuri S; Singh, Yogesh; Zelenak, Christine; Ghashghaeinia, Mehrdad; Gu, Shuchen; Jilani, Kashif; Lupescu, Adrian; Reyskens, Kathleen M S E; Ackermann, Teresa F; Föller, Michael; Schleicher, Erwin; Sheffield, William P; Arthur, J Simon C; Lang, Florian; Qadri, Syed M

    2015-01-01

    The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk(-/-)) and corresponding wild-type mice (msk(+/+)). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk(-/-) and msk(+/+) mice, but reticulocyte count was significantly increased in msk(-/-) mice. Cell membrane PS exposure was similar in untreated msk(-/-) and msk(+/+) erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk(-/-) erythrocytes than in msk(+/+) erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk(-/-) erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk(-/-) mice. The spleens from msk(-/-) mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk(+/+) mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice. PMID:26611568

  12. Folic acid-functionalized up-conversion nanoparticles: toxicity studies in vivo and in vitro and targeted imaging applications

    NASA Astrophysics Data System (ADS)

    Sun, Lining; Wei, Zuwu; Chen, Haige; Liu, Jinliang; Guo, Jianjian; Cao, Ming; Wen, Tieqiao; Shi, Liyi

    2014-07-01

    Folate receptors (FRs) are overexpressed on a variety of human cancer cells and tissues, including cancers of the breast, ovaries, endometrium, and brain. This over-expression of FRs can be used to target folate-linked imaging specifically to FR-expressing tumors. Fluorescence is emerging as a powerful new modality for molecular imaging in both the diagnosis and treatment of disease. Combining innovative molecular biology and chemistry, we prepared three kinds of folate-targeted up-conversion nanoparticles as imaging agents (UCNC-FA: UCNC-Er-FA, UCNC-Tm-FA, and UCNC-Er,Tm-FA). In vivo and in vitro toxicity studies showed that these nanoparticles have both good biocompatibility and low toxicity. Moreover, the up-conversion luminescence imaging indicated that they have good targeting to HeLa cells and can therefore serve as potential fluorescent contrast agents.Folate receptors (FRs) are overexpressed on a variety of human cancer cells and tissues, including cancers of the breast, ovaries, endometrium, and brain. This over-expression of FRs can be used to target folate-linked imaging specifically to FR-expressing tumors. Fluorescence is emerging as a powerful new modality for molecular imaging in both the diagnosis and treatment of disease. Combining innovative molecular biology and chemistry, we prepared three kinds of folate-targeted up-conversion nanoparticles as imaging agents (UCNC-FA: UCNC-Er-FA, UCNC-Tm-FA, and UCNC-Er,Tm-FA). In vivo and in vitro toxicity studies showed that these nanoparticles have both good biocompatibility and low toxicity. Moreover, the up-conversion luminescence imaging indicated that they have good targeting to HeLa cells and can therefore serve as potential fluorescent contrast agents. Electronic supplementary information (ESI) available: Up-conversion luminescence spectra of UCNC-Er and UCNC-Er-FA, UCNC-Tm and UCNC-Tm-FA. Confocal luminescence imaging data collected as a series along the Z optical axis. See DOI: 10.1039/c4nr02312a

  13. Function prediction and analysis of mycobacterium tuberculosis hypothetical proteins.

    PubMed

    Mazandu, Gaston K; Mulder, Nicola J

    2012-01-01

    High-throughput biology technologies have yielded complete genome sequences and functional genomics data for several organisms, including crucial microbial pathogens of humans, animals and plants. However, up to 50% of genes within a genome are often labeled "unknown", "uncharacterized" or "hypothetical", limiting our understanding of virulence and pathogenicity of these organisms. Even though biological functions of proteins encoded by these genes are not known, many of them have been predicted to be involved in key processes in these organisms. In particular, for Mycobacterium tuberculosis, some of these "hypothetical" proteins, for example those belonging to the Pro-Glu or Pro-Pro-Glu (PE/PPE) family, have been suspected to play a crucial role in the intracellular lifestyle of this pathogen, and may contribute to its survival in different environments. We have generated a functional interaction network for Mycobacterium tuberculosis proteins and used this to predict functions for many of its hypothetical proteins. Here we performed functional enrichment analysis of these proteins based on their predicted biological functions to identify annotations that are statistically relevant, and analysed and compared network properties of hypothetical proteins to the known proteins. From the statistically significant annotations and network information, we have tried to derive biologically meaningful annotations related to infection and disease. This quantitative analysis provides an overview of the functional contributions of Mycobacterium tuberculosis "hypothetical" proteins to many basic cellular functions, including its adaptability in the host system and its ability to evade the host immune response. PMID:22837694

  14. Transcriptome-wide Analysis Reveals Hallmarks of Human Intestine Development and Maturation In Vitro and In Vivo

    PubMed Central

    Finkbeiner, Stacy R.; Hill, David R.; Altheim, Christopher H.; Dedhia, Priya H.; Taylor, Matthew J.; Tsai, Yu-Hwai; Chin, Alana M.; Mahe, Maxime M.; Watson, Carey L.; Freeman, Jennifer J.; Nattiv, Roy; Thomson, Matthew; Klein, Ophir D.; Shroyer, Noah F.; Helmrath, Michael A.; Teitelbaum, Daniel H.; Dempsey, Peter J.; Spence, Jason R.

    2015-01-01

    Summary Human intestinal organoids (HIOs) are a tissue culture model in which small intestine-like tissue is generated from pluripotent stem cells. By carrying out unsupervised hierarchical clustering of RNA-sequencing data, we demonstrate that HIOs most closely resemble human fetal intestine. We observed that genes involved in digestive tract development are enriched in both fetal intestine and HIOs compared to adult tissue, whereas genes related to digestive function and Paneth cell host defense are expressed at higher levels in adult intestine. Our study also revealed that the intestinal stem cell marker OLFM4 is expressed at very low levels in fetal intestine and in HIOs, but is robust in adult crypts. We validated our findings using in vivo transplantation to show that HIOs become more adult-like after transplantation. Our study emphasizes important maturation events that occur in the intestine during human development and demonstrates that HIOs can be used to model fetal-to-adult maturation. PMID:26050928

  15. In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector

    SciTech Connect

    Wang Shengpeng; Guo Tingqing; Guo Xiuyang; Huang Junting; Lu Changde . E-mail: cdlu@sibs.ac.cn

    2006-03-24

    In order to investigate the functional signal peptide of silkworm fibroin heavy chain (FibH) and the effect of N- and C-terminal parts of FibH on the secretion of FibH in vivo, N- and C-terminal segments of fibh gene were fused with enhanced green fluorescent protein (EGFP) gene. The fused gene was then introduced into silkworm larvae and expressed in silk gland using recombinant AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) as vector. The fluorescence of EGFP was observed with fluorescence microscope. FibH-EGFP fusion proteins extracted from silk gland were analyzed by Western blot. Results showed that the two alpha helices within N-terminal 163 amino acid residues and the C-terminal 61 amino acid residues were not necessary for cleavage of signal peptide and secretion of the fusion protein into silk gland. Then the C-terminal 61 amino acid residues were substituted with a His-tag in the fusion protein to facilitate the purification. N-terminal sequencing of the purified protein showed that the signal cleavage site is between position 21 and 22 amino acid residues.

  16. The Necessity of Functional Analysis for Space Exploration Programs

    NASA Technical Reports Server (NTRS)

    Morris, A. Terry; Breidenthal, Julian C.

    2011-01-01

    As NASA moves toward expanded commercial spaceflight within its human exploration capability, there is increased emphasis on how to allocate responsibilities between government and commercial organizations to achieve coordinated program objectives. The practice of program-level functional analysis offers an opportunity for improved understanding of collaborative functions among heterogeneous partners. Functional analysis is contrasted with the physical analysis more commonly done at the program level, and is shown to provide theoretical performance, risk, and safety advantages beneficial to a government-commercial partnership. Performance advantages include faster convergence to acceptable system solutions; discovery of superior solutions with higher commonality, greater simplicity and greater parallelism by substituting functional for physical redundancy to achieve robustness and safety goals; and greater organizational cohesion around program objectives. Risk advantages include avoidance of rework by revelation of some kinds of architectural and contractual mismatches before systems are specified, designed, constructed, or integrated; avoidance of cost and schedule growth by more complete and precise specifications of cost and schedule estimates; and higher likelihood of successful integration on the first try. Safety advantages include effective delineation of must-work and must-not-work functions for integrated hazard analysis, the ability to formally demonstrate completeness of safety analyses, and provably correct logic for certification of flight readiness. The key mechanism for realizing these benefits is the development of an inter-functional architecture at the program level, which reveals relationships between top-level system requirements that would otherwise be invisible using only a physical architecture. This paper describes the advantages and pitfalls of functional analysis as a means of coordinating the actions of large heterogeneous organizations for space exploration programs.

  17. Anatomical and Functional Images of in vitro and in vivo Tissues by NIR Time-domain Diffuse Optical Tomography

    NASA Astrophysics Data System (ADS)

    Zhao, Huijuan; Gao, Feng; Tanikawa, Yukari; Homma, Kazuhiro; Onodera, Yoichi; Yamada, Yukio

    Near infra-red (NIR) diffuse optical tomography (DOT) has gained much attention and it will be clinically applied to imaging breast, neonatal head, and the hemodynamics of the brain because of its noninvasiveness and deep penetration in biological tissue. Prior to achieving the imaging of infant brain using DOT, the developed methodologies need to be experimentally justified by imaging some real organs with simpler structures. Here we report our results of an in vitro chicken leg and an in vivo exercising human forearm from the data measured by a multi-channel time-resolved NIR system. Tomographic images were reconstructed by a two-dimensional image reconstruction algorithm based on a modified generalized pulse spectrum technique for simultaneous reconstruction of the µa and µs´. The absolute µa- and µs´-images revealed the inner structures of the chicken leg and the forearm, where the bones were clearly distinguished from the muscle. The ?µa-images showed the blood volume changes during the forearm exercise, proving that the system and the image reconstruction algorithm could potentially be used for imaging not only the anatomic structure but also the hemodynamics in neonatal heads.

  18. Tongxinluo (TXL), a Traditional Chinese Medicinal Compound, Improves Endothelial Function After Chronic Hypoxia Both In Vivo and In Vitro

    PubMed Central

    Zheng, Cui-Ying; Song, Li-Li; Wen, Jin-Kun; Li, Li-Min; Guo, Zong-Wei; Zhou, Pei-Pei; Wang, Chang; Li, Yong-Hui; Ma, Dong

    2015-01-01

    Abstract: Vascular injury after chronic hypoxia leads to endothelial injury and structural damage to tight junctions (TJs), thereby resulting in a variety of cardiovascular diseases. Thus, attenuating hypoxia-induced damage has great significance for the prevention and treatment of cardiovascular disease. The aim of this study was to investigate whether the endothelial protection conferred by tongxinluo (TXL), a traditional Chinese medicinal compound, is related to its regulation of TJ protein expression. In vivo, we found that TXL could promote hypoxia-induced angiogenesis in lung and liver tissue. In vitro, we found that CoCl2 treatment significantly reduced the expression of the TJ proteins occludin, claudin-1, VE-cadherin, and beta-catenin in cultured human cardiac microvascular endothelial cells. TXL pretreatment abrogated the CoCl2-induced downregulation of these TJ proteins. Conversely, overexpression of Krüppel-like factor 4 (KLF4) inhibited the expression of TJ proteins in human cardiac microvascular endothelial cells, an effect that was reversed by TXL pretreatment. Further experiments showed that TXL could promote endothelial cell proliferation by increasing KLF4 phosphorylation, thereby reversing the effect of KLF4 on the expression of TJ proteins. These findings provide a new molecular mechanism for the TXL-induced increase in TJ protein expression. PMID:26065642

  19. A genetic system to assess in vivo the functions of histones and histone modifications in higher eukaryotes

    PubMed Central

    Günesdogan, Ufuk; Jäckle, Herbert; Herzig, Alf

    2010-01-01

    Despite the fundamental role of canonical histones in nucleosome structure, there is no experimental system for higher eukaryotes in which basic questions about histone function can be directly addressed. We developed a new genetic tool for Drosophila melanogaster in which the canonical histone complement can be replaced with multiple copies of experimentally modified histone transgenes. This new histone-replacement system provides a well-defined and direct cellular assay system for histone function with which to critically test models in chromatin biology dealing with chromatin assembly, variant histone functions and the biological significance of distinct histone modifications in a multicellular organism. PMID:20814422

  20. Co-Localized or Randomly Distributed? Pair Cross Correlation of In Vivo Grown Subgingival Biofilm Bacteria Quantified by Digital Image Analysis

    PubMed Central

    Lux, Renate; Riep, Birgit; Kikhney, Judith; Friedmann, Anton; Wolinsky, Lawrence E.; Göbel, Ulf B.; Daims, Holger; Moter, Annette

    2012-01-01

    The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 µm) with a pronounced peak at 1.5 µm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for “as-yet-uncultured” phylotypes which cannot be characterized in vitro. PMID:22655057

  1. Characterization of the alpha-gamma and alpha-beta complex: evidence for an in vivo functional role of alpha-crystallin as a molecular chaperone

    NASA Technical Reports Server (NTRS)

    Boyle, D.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Previous studies have demonstrated that in vitro, alpha-crystallin can protect other lens proteins against extensive denaturation and aggregation. The mechanism of this protection involves preferential binding of the partially denatured protein to a central region of the native alpha-crystallin complex. To test whether a similar phenomenon might occur in vivo, a high molecular weight aggregate (HMWA) fraction was isolated from the aged bovine lens. Negative staining of this preparation revealed the presence of particles of 13-14 nm diameter, characteristic of alpha-crystallin. Immunolocalization of the same particles using antiserum specific for gamma- and beta-crystallins demonstrated preferential binding of these crystallins to the central region of the alpha-crystallin complex. Together, these results provide evidence that in the intact lens, the alpha-crystallins are functionally important molecular chaperones.

  2. Structural Dynamics of Synapses in Vivo Correlate with Functional Changes during Experience-Dependent Plasticity in Visual Cortex

    E-print Network

    Tropea, Daniela

    The impact of activity on neuronal circuitry is complex, involving both functional and structural changes whose interaction is largely unknown. We have used optical imaging of mouse visual cortex responses and two-photon ...

  3. Functional Identification of Tumor Suppressor Genes Through an in vivo RNA Interference Screen in a Mouse Lymphoma Model

    E-print Network

    Bric, Anka

    Short hairpin RNAs (shRNAs) capable of stably suppressing gene function by RNA interference (RNAi) can mimic tumor-suppressor-gene loss in mice. By selecting for shRNAs capable of accelerating lymphomagenesis in a ...

  4. In vitro and in vivo analysis of pro- and anti-inflammatory effects of weak and strong contact allergens.

    PubMed

    Lass, Christian; Merfort, Irmgard; Martin, Stefan F

    2010-11-01

    Inflammation is a crucial step in the development of allergic contact dermatitis. The primary contact with chemical allergens, called sensitization, and the secondary contact, called elicitation, result in an inflammatory response in the skin. The ability of contact allergens to induce allergic contact dermatitis correlates to a great extent with their inflammatory potential. Therefore, the analysis of the sensitizing potential of a putative contact allergen should include the examination of its ability and potency to cause an inflammation. In this study, we examined the inflammatory potential of different weak contact allergens and of the strong sensitizer 2,4,6-trinitrochlorobenzene (TNCB) in vitro and in vivo using the contact hypersensitivity model, the mouse model for allergic contact dermatitis. Cytokine induction was analysed by PCR and ELISA to determine mRNA and protein levels, respectively. Inflammation-dependent recruitment of skin-homing effector T cells was measured in correlation with the other methods. We show that the sensitizing potential of a contact allergen correlates with the strength of the inflammatory response. The different methods used gave similar results. Quantitative cytokine profiling may be used to determine the sensitizing potential of chemicals for hazard identification and risk assessment. PMID:20701630

  5. In vivo evaluation of the elastic anisotropy of the human Achilles tendon using shear wave dispersion analysis

    NASA Astrophysics Data System (ADS)

    Brum, J.; Bernal, M.; Gennisson, J. L.; Tanter, M.

    2014-02-01

    Non-invasive evaluation of the Achilles tendon elastic properties may enhance diagnosis of tendon injury and the assessment of recovery treatments. Shear wave elastography has shown to be a powerful tool to estimate tissue mechanical properties. However, its applicability to quantitatively evaluate tendon stiffness is limited by the understanding of the physics on the shear wave propagation in such a complex medium. First, tendon tissue is transverse isotropic. Second, tendons are characterized by a marked stiffness in the 400 to 1300 kPa range (i.e. fast shear waves). Hence, the shear wavelengths are greater than the tendon thickness leading to guided wave propagation. Thus, to better understand shear wave propagation in tendons and consequently to properly estimate its mechanical properties, a dispersion analysis is required. In this study, shear wave velocity dispersion was measured in vivo in ten Achilles tendons parallel and perpendicular to the tendon fibre orientation. By modelling the tendon as a transverse isotropic viscoelastic plate immersed in fluid it was possible to fully describe the experimental data (deviation<1.4%). We show that parallel to fibres the shear wave velocity dispersion is not influenced by viscosity, while it is perpendicularly to fibres. Elasticity (found to be in the range from 473 to 1537 kPa) and viscosity (found to be in the range from 1.7 to 4 Pa.s) values were retrieved from the model in good agreement with reported results.

  6. Ultrastructural Analysis of In Vivo Hypoglycemiant Effect of Two Polyoxometalates in Rats with Streptozotocin-Induced Diabetes.

    PubMed

    Bâlici, ?tefana; Wankeu-Nya, Modeste; Rusu, Dan; Nicula, Gheorghe Z; Rusu, Mariana; Florea, Adrian; Matei, Horea

    2015-10-01

    Two polyoxometalates (POMs), synthesized through a self-assembling method, were used in the treatment of streptozotocin (STZ)-induced diabetic rats. One of these nanocompounds [tris(vanadyl)-substituted tungsto-antimonate(III)-anions-POM1] was previously described in the literature, whereas the second [tris-butyltin-21-tungsto-9-antimonate(III)-anions-POM2], was prepared by us based on our original formula. In rats with STZ-induced diabetes treated with POMs (up to a cumulative dose of 4 mg/kg bodyweight at the end of the treatments), statistically significant reduced levels of blood glucose were measured after 3 weeks, as compared with the diabetic control groups (DCGs). Ultrastructural analysis of pancreatic ?-cells (including the mean diameter of secretory vesicles and of their insulin granules) in the treated diabetic rats proved the POMs contribute to limitation of cellular degeneration triggered by STZ, as well as to the presence of increased amounts of insulin-containing vesicles as compared with the DCG. The two POMs also showed hepatoprotective properties when ultrastructural aspects of hepatocytes in the experimental groups of rats were studied. Based on our in vivo studies, we concluded that the two POMs tested achieved hypoglycemiant effects by preventing STZ-triggered apoptosis of pancreatic ?-cells and stimulation of insulin synthesis. PMID:26343528

  7. In vivo pharmacokinetic analysis for fluorescently labeled RGD peptide targeted to the ?v?3 integrin in Kaposi"s sarcoma

    NASA Astrophysics Data System (ADS)

    Kwon, Sunkuk; Ke, Shi; Houston, Jessica P.; Wang, Wei; Wu, Qingping; Li, Chun; Sevick Muraca, Eva M.

    2005-04-01

    The dose dependence of near-infrared (NIR) fluorescent labeled RGD peptide targeted to the ?v?3 integrin was assessed from xenografts bearing a subcutaneous human Kaposi"s sarcoma (KS1767) with dynamic NIR fluorescence optical imaging. The three-compartment pharmacokinetic (PK) model was used to determine PK parameters from fluorescence images acquired with an intensified charge-coupled device (ICCD) system. Dynamic imaging of Kaposi"s sarcoma bearing animals was conducted with i.v. administration of Cy5.5-c(KRGDf) at doses of 0.75 to 6 nmol/animal and at the doses of 300 or 600 nmol of c(KRGDf) administered 1 hour before the injection of 3 nmol dose of the conjugate. The results show early and rapid uptake of Cy5.5-c(KRGDf), which was mediated by the administration of c(KRGDf) 1 hour before administration at the conjugate agent. From the results we found a linear increase in PK uptake rates at doses of 0.75 to 1.5 nmol, reflecting unsaturated binding to the integrin receptor. However, the results show the dose independence at large dose amounts from 3 to 6 nmol per animal. The effects of cancer treatments as well as diagnostics may be evaluated by in vivo PK analysis with NIR fluorescence optical imaging.

  8. Construction of Training Sets for Valid Calibration of in Vivo Cyclic Voltammetric Data by Principal Component Analysis.

    PubMed

    Rodeberg, Nathan T; Johnson, Justin A; Cameron, Courtney M; Saddoris, Michael P; Carelli, Regina M; Wightman, R Mark

    2015-11-17

    Principal component regression, a multivariate calibration technique, is an invaluable tool for the analysis of voltammetric data collected in vivo with acutely implanted microelectrodes. This method utilizes training sets to separate cyclic voltammograms into contributions from multiple electroactive species. The introduction of chronically implanted microelectrodes permits longitudinal measurements at the same electrode and brain location over multiple recordings. The reliability of these measurements depends on a consistent calibration methodology. One published approach has been the use of training sets built with data from separate electrodes and ani