Science.gov

Sample records for vivo functional analysis

  1. Thermal analysis of laser interstitial thermotherapy in ex vivo fibro-fatty tissue using exponential functions

    NASA Astrophysics Data System (ADS)

    Salas, Nelson, Jr.; Manns, Fabrice; Milne, Peter J.; Denham, David B.; Minhaj, Ahmed M.; Parel, Jean-Marie; Robinson, David S.

    2004-05-01

    A therapeutic procedure to treat small, surface breast tumours up to 10 mm in radius plus a 5 mm margin of healthy, surrounding tissue using laser interstitial thermotherapy (LITT) is currently being investigated. The purpose of this study is to analyse and model the thermal and coagulative response of ex vivo fibro-fatty tissue, a model for breast tissue, during experimental laser interstitial thermotherapy at 980 nm. Laser radiation at 980 nm was delivered interstitially through a diffusing tip optical fibre inserted into a fibro-fatty tissue model to produce controlled heating at powers ranging from 3.2 to 8.0 W. Tissue temperature was measured with thermocouples placed at 15 positions around the fibre. The induced coagulation zone was measured on gross anatomical sections. Thermal analysis indicates that a finite sum of exponential functions is an approximate solution to the heat conduction equation that more accurately predicts the time-temperature dependence in tissue prior to carbonization (T < 100 °C) during LITT than the traditional model using a single exponential function. Analysis of the ellipsoid coagulation volume induced in tissue indicates that the 980 nm wavelength does not penetrate deep enough in fibro-fatty tissue to produce a desired 30 mm diameter (14.1 × 103 mm3) coagulation volume without unwanted tissue liquefaction and carbonization.

  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 β-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model. PMID:21074529

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

    NASA Astrophysics Data System (ADS)

    Zhao, Dong

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

  4. Spectroscopic analysis of titanium surface functional groups under various surface modification and their behaviors in vitro and in vivo.

    PubMed

    Lu, Xiong; Wang, Yingbo; Yang, Xiudong; Zhang, Qiyi; Zhao, Zhanfeng; Weng, Lu-Tao; Leng, Yang

    2008-02-01

    In the present study, surface functional groups of titanium surfaces gone through different treatments, including acid etched treatment (AE), nitric acid treatment (NT), heat treatment (HT), and alkali treatment (AT), and their behaviors in vitro and in vivo was thoroughly studied by spectroscopic analysis. In vitro and in vivo results revealed that the rank of bioactivity of various surfaces was AE < NT < HT < AT. XPS analysis indicated that AT greatly increased the OH group concentration on the titanium surface whereas HT reduced the OH group concentration. Thus, OH group difference could not be a good explanation of bioactivity difference. On the other hand, ToF-SIMS analysis demonstrated the TiOH+/Ti+ ratios of various surfaces correlated well with the bioactivity and the surface energies, which implied that Ti-OH could play an important role in the bioactivity. This detail investigation of the relationship between surface functional groups and surface bioactivity could help us to broaden the knowledge about the mechanism of bioactivity and to design next generation bioactive materials. PMID:17618503

  5. An ES cell system for rapid, spatial and temporal analysis of gene function in vitro and in vivo

    PubMed Central

    Mao, Junhao; Barrow, Jeffery; McMahon, Jill; Vaughan, Joe; McMahon, Andrew P.

    2005-01-01

    We describe a versatile genetic system for rapid analysis of mammalian gene function. In this, loss of reporter activity in a novel embryonic stem (ES) cell line enables rapid identification of targeting to the ubiquitously expressed Rosa26 locus. Subsequent regulation of gene activity is governed by a dual regulatory strategy utilizing two drugs, Tamoxifen and Doxycycline. To illustrate this approach, a dominant allele of Smoothened was introduced into this cell line, enabling regulated activation of Hedgehog signaling. By coupling Cre-loxP dependent activation with tetracycline dependent transcription in a single allele, we established a conditional method to control Smoothened activity and neural progenitor specification in differentiating ES cells in vitro and in chimeric embryos in vivo When crossed to an appropriate Cre driver strain, gene activity can also be temporally regulated within a specific cell lineage. This platform will facilitate rapid analysis of gene function in the mouse. PMID:16221970

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2007-02-01

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

  8. Specific Schistosoma mansoni rat T cell clones. I. Generation and functional analysis in vitro and in vivo.

    PubMed

    Pestel, J; Dissous, C; Dessaint, J P; Louis, J; Engers, H; Capron, A

    1985-06-01

    In an attempt to determine the role of schistosome-specific T cells in the immune mechanisms developed during schistosomiasis, Schistosoma mansoni-specific T cells and clones were generated in vitro and some of their functions analyzed in vitro and in vivo in the fischer rat model. The data presented here can be summarized as follows: a) Lymph node cells (LNC) from rats primed with the excretory/secretory antigens-incubation products (IPSm) of adult worms proliferate in vitro only in response to the homologous schistosome antigens and not to unrelated antigens (Ag) such as ovalbumin (OVA) or Dipetalonema viteae and Fasciola hepatica parasite extracts. b) After in vitro restimulation of the primed LNC population with IPSm in the presence of antigen-presenting cells (APC) and maintenance in IL 2-containing medium, the frequency of IPSm-specific T cells is increased and the T cells can be restimulated only in the presence of APC possessing the same major histocompatibility complex (MHC) antigens. c) Following appropriate limiting dilution assays (LDA) (1 cell/well), 10 IPSm-specific T cell clones were obtained, and two of four maintained in culture were tested for their helper activity because they expressed only the W3/13+ W3/25+ surface phenotypes. d) The two highly proliferating IPSm-specific T cell clones (G5 and E23) exhibit an IPSm-dependent helper activity, as shown by the increase in IgG production by IPSm-primed B cells. e) IPSm-T cell clone (G5) as well as IPSm-T cell lines when injected in S. mansoni-infested rats can exert an in vivo helper activity, which is characterized by an accelerated production of IgG antibodies specific for the previously identified 30 to 40 kilodaltons (kd) schistosomula surface antigens (Ag). As recent studies have demonstrated that rat monoclonal antibodies recognize some incubation products of adult S. mansoni as well as one of the 30 to 40 kd schistosomula surface antigens, and taking into account the fact that the T cell

  9. 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 ι/λ and ζ 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 PKCι in mouse development we designed PKCι deletion mutants using the gene targeting approach. We show that the deletion of PKCι, 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 PKCι, tight junctions and apico-basal polarity were still established. Finally, our study points to a non-redundant PKCι function at E9.5, since expression of PKCζ 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 PKCι is crucial for mouse embryogenesis but is dispensable for the establishment of polarity and tight junction formation. We present a compensatory function of PKCζ at E7.5, rescuing the phenotype. Furthermore, this study indicates at least one specific, yet unknown, PKCι function that cannot be compensated by the overexpression of PKCζ at E9.5. PMID:23690951

  10. 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

  11. In Vivo Performance of a Novel Fluorinated Magnetic Resonance Imaging Agent for Functional Analysis of Bile Acid Transport

    PubMed Central

    2015-01-01

    A novel trifluorinated cholic acid derivative, CA-lys-TFA, was designed and synthesized for use as a tool to measure bile acid transport noninvasively using magnetic resonance imaging (MRI). In the present study, the in vivo performance of CA-lys-TFA for measuring bile acid transport by MRI was investigated in mice. Gallbladder CA-lys-TFA content was quantified using MRI and liquid chromatography/tandem mass spectrometry. Results in wild-type (WT) C57BL/6J mice were compared to those in mice lacking expression of Asbt, the ileal bile acid transporter. 19F signals emanating from the gallbladders of WT mice 7 h after oral gavage with 150 mg/kg CA-lys-TFA were reproducibly detected by MRI. Asbt-deficient mice administered the same dose had undetectable 19F signals by MRI, and gallbladder bile CA-lys-TFA levels were 30-fold lower compared to WT animals. To our knowledge, this represents the first report of in vivo imaging of an orally absorbed drug using 19F MRI. Fluorinated bile acid analogues have potential as tools to measure and detect abnormal bile acid transport by MRI. PMID:24708306

  12. Proteomic analysis of the function of spot in Helicobacter pylori anti-oxidative stress in vitro and colonization in vivo.

    PubMed

    Sun, Yundong; Li, Xinpeng; Li, Wen; Zhao, Min; Wang, Lixiang; Liu, Shili; Zeng, Jiping; Liu, Zhifang; Jia, Jihui

    2012-11-01

    As a microaerobe, Helicobacter pylori employs the global regulator SpoT for defending against oxidative stress in vitro. However, the mechanisms how SpoT affects bacterial gene expression is still unknown. Moreover, the function of SpoT in H. pylori colonization in the host is remaining undetermined. To explore the functions of the SpoT in H. pylori pathogenesis, we constructed H. pylori 26695 spoT-deficient mutant (ΔspoT). While grown in ambient atmosphere, protein expression profile of the ΔspoT was analyzed with 2D gel electrophoresis and real-time PCR. Compared to the wild type, the spoT-deficient strain downregulated its transcription of the oxidative-induced genes, as well as the genes responsible for protein degradation and that related to energy metabolism. Meanwhile, the colonization ability of ΔspoT strains in Mongolian gerbil was tested, the results demonstrated a decayed colonization in the mouse stomach with ΔspoT than the wild type. As a matter of facts, the AGS cells infected with the ΔspoT strains excreted increased level of the gastric inflammation cytokines IL-8, and the ΔspoT strains showed poor survival ability when treated with reactive oxygen stress (sodium nitroprusside). The elevated capacity of stimulating cytokines and fragility to reactive oxygen stress may be contribute to decreased colonization of the spoT-deficient mutant in the mouse stomach. Conclusively, we speculate that spoT is a key regulator of the genes for H. pylori spreading in the air and colonization in host stomach. PMID:22678710

  13. 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 ...

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

    PubMed

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

    2012-12-01

    In addition to their well-accepted role as critical effector cells in anaphylaxis and other acute IgE-mediated allergic reactions, mast cells (MCs) have been implicated in a wide variety of processes that contribute to disease or help to maintain health. Although some of these roles were first suggested by analyses of MC products or functions in vitro, it is critical to determine whether, and under which circumstances, such potential roles actually can be performed by MCs in vivo. This review discusses recent advances in the development and analysis of mouse models to investigate the roles of MCs and MC-associated products during biological responses in vivo, and comments on some of the similarities and differences in the results obtained with these newer versus older models of MC deficiency. PMID:23127755

  15. Functional analysis in vivo of engineered valved venous conduit with decellularized matrix and two bone marrow-derived progenitors in sheep.

    PubMed

    Yuan, Jian-Ming; Xiong, Shao-Hu; Liu, Zhen; Wen, Yu; Dang, Rui-Shan; Shen, Man-Ru; Zhang, Yong-Zhen; Zhang, Xi; Yang, Xiang-Qun; Zhang, Chuan-Sen

    2016-07-01

    Tissue engineering has been considered a promising approach for creating grafts to replace autologous venous valves. Here, ovine bone marrow-derived endothelial progenitor cells (EPCs) and multipotent adult progenitor cells (MAPCs) were harvested and then loaded into decellularized venous matrix to create tissue-engineered (TE) valved vein. Subsequently, the ovine femoral veins containing the valve were removed and replaced by TE grafts or acellular matrix only. The morphology and function were analysed for up to 1 year by ultrasonography, angiography, H&E staining and scanning electron microscopy (SEM). The differentiation of seeded cells was traced immunofluorochemically. The results showed that decellularized venous matrix could initially and feebly attract endogenous cells, but failed afterwards and were insufficient to restore valve function. On the contrary, the seeded cells differentiated into endothelial cells (ECs) in vivo and formed a monolayer endothelium, and smooth muscle cells within the scaffold therefore produced TE grafts comparable to the native vein valve. This TE graft remained patent and sufficient after implantation into the venous circuit of the ovine lower extremity for at least 6 months. Unfortunately, cells seeded on the luminal surface and both sides of the leaflets lost their biological functions at 12 months, resulting in thrombosis formation and leading to complete occlusion of the TE grafts and impotent venous valves. These findings suggest that this TE valved venous conduit can function physiologically in vivo in the medium term. Before translating this TE venous valve into clinical practice, the durability should be improved and thrombogenicity should be suppressed. Copyright © 2016 John Wiley & Sons, Ltd. PMID:23904287

  16. GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

    PubMed Central

    Greenberg, Anthony J.; Schedl, Paul

    2001-01-01

    The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo. PMID:11713290

  17. 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

  18. Identification of the determinants of tRNA function and susceptibility to rapid tRNA decay by high-throughput in vivo analysis

    PubMed Central

    Guy, Michael P.; Young, David L.; Payea, Matthew J.; Zhang, Xiaoju; Kon, Yoshiko; Dean, Kimberly M.; Grayhack, Elizabeth J.; Mathews, David H.; Fields, Stanley

    2014-01-01

    Sequence variation in tRNA genes influences the structure, modification, and stability of tRNA; affects translation fidelity; impacts the activity of numerous isodecoders in metazoans; and leads to human diseases. To comprehensively define the effects of sequence variation on tRNA function, we developed a high-throughput in vivo screen to quantify the activity of a model tRNA, the nonsense suppressor SUP4oc of Saccharomyces cerevisiae. Using a highly sensitive fluorescent reporter gene with an ochre mutation, fluorescence-activated cell sorting of a library of SUP4oc mutant yeast strains, and deep sequencing, we scored 25,491 variants. Unexpectedly, SUP4oc tolerates numerous sequence variations, accommodates slippage in tertiary and secondary interactions, and exhibits genetic interactions that suggest an alternative functional tRNA conformation. Furthermore, we used this methodology to define tRNA variants subject to rapid tRNA decay (RTD). Even though RTD normally degrades tRNAs with exposed 5′ ends, mutations that sensitize SUP4oc to RTD were found to be located throughout the sequence, including the anti-codon stem. Thus, the integrity of the entire tRNA molecule is under surveillance by cellular quality control machinery. This approach to assess activity at high throughput is widely applicable to many problems in tRNA biology. PMID:25085423

  19. 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

  20. Construction of an in vivo system for functional analysis of the genes involved in sex pheromone production in the silkmoth, Bombyx mori.

    PubMed

    Moto, Ken-Ichi; Matsumoto, Shogo

    2012-01-01

    Moths produce species-specific sex pheromones to attract conspecific mates. The biochemical processes that comprise sex pheromone biosynthesis are precisely regulated and a number of gene products are involved in this biosynthesis and regulation. In recent years, at least 300 EST clones have been isolated from Bombyx mori pheromone gland (PG) specific cDNA libraries with some of those clones [i.e., B. mori PG-specific desaturase 1 (Bmpgdesat1), PG-specific fatty acyl reductase, PG-specific acyl-CoA-binding protein, B. mori fatty acid transport protein, B. mori lipid storage droplet protein-1] characterized and demonstrated to play a role in sex pheromone production. However, most of the EST clones have yet to be fully characterized and identified. To develop an efficient system for analyzing sex pheromone production-related genes, we investigated the feasibility of a novel gene analysis system using the upstream region of Bmpgdesat1 that should contain a PG-specific gene promoter in conjunction with piggyBac vector-mediated germ line transformation. As a result, we have been able to obtain expression of our reporter gene (enhanced green fluorescent protein) in the PG but not in other tissues of transgenic B. mori. Current results indicate that we have successfully constructed a novel in vivo gene analysis system for sex pheromone production in B. mori. PMID:22649415

  1. Spectroscopy analysis of tissues in vivo

    NASA Astrophysics Data System (ADS)

    Loschenov, Victor B.; Poleshkin, P. V.; Stratonnikov, Alexander A.; Torshina, Nadezgda L.

    1995-01-01

    The spectral analysis of biological tissues in vivo is widely used in various fields particularly in medical diagnostics and therapy control. Great possibilities of spectral tissue analysis exist to be realized in the future. Among them are the complete non-invasive clinical blood analysis with evaluation of, for example, sugar concentration in blood; the evaluation of chemical state and localization on subcell level of various drugs binded with biological structures. These facts were shown to affect drastically the drug therapeutic activity. The main advantage of spectral analysis of tissues in vivo is its noninvasivity. This allows one to get information about tissue condition without affecting the dynamic of various biological processes. Another advantage of optical tissue analysis is the possibility to process data in real time and to control parameters of therapy process according to information acquired. For example the in situ analysis of photosensitizer concentration and its chemical state during photodynamic therapy makes it possible to correct the laser irradiation intensity (the photobleaching of photosensitizer requires the decrease in laser intensity).

  2. In vivo activation and functions of the protease factor XII.

    PubMed

    Björkqvist, Jenny; Nickel, Katrin F; Stavrou, Evi; Renné, Thomas

    2014-11-01

    Combinations of proinflammatory and procoagulant reactions are the unifying principle for a variety of disorders affecting the cardiovascular system. Factor XII (FXII, Hageman factor) is a plasma protease that initiates the contact system. The biochemistry of the contact system in vitro is well understood; however, its in vivo functions are just beginning to emerge. The current review concentrates on activators and functions of the FXII-driven contact system in vivo. Elucidating its physiologic activities offers the exciting opportunity to develop strategies for the safe interference with both thrombotic and inflammatory diseases. PMID:25187064

  3. In Vivo Analysis of Saccharomyces Cerevisiae Cox2 mRNA 5'-Untranslated Leader Functions in Mitochondrial Translation Initiation and Translational Activation

    PubMed Central

    Dunstan, H. M.; Green-Willms, N. S.; Fox, T. D.

    1997-01-01

    We have used mutational and revertant analysis to study the elements of the 54-nucleotide COX2 5'-untranslated leader involved in translation initiation in yeast mitochondria and in activation by the COX2 translational activator, Pet111p. We generated a collection of mutants with substitutions spanning the entire COX2 5'-UTL by in vitro mutagenesis followed by mitochondrial transformation and gene replacement. The phenotypes of these mutants delimit a 31-nucleotide segment, from -16 to -46, that contains several short sequence elements necessary for COX2 5'-UTL function in translation. The sequences from -16 to -47 were shown to be partially sufficient to promote translation in a foreign context. Analysis of revertants of both the series of linker-scanning alleles and two short deletion/insertion alleles has refined the positions of several possible functional elements of the COX2 5'-untranslated leader, including a putative RNA stem-loop structure that functionally interacts with Pet111p and an octanucleotide sequence present in all S. cerevisiae mitochondrial mRNA 5'-UTLs that is a potential rRNA binding site. PMID:9286670

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

    PubMed

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

    2008-01-01

    There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine), obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity. PMID:18493600

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

    PubMed Central

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

    2008-01-01

    There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine), obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity. PMID:18493600

  6. 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

  7. Detection of Tight Junction Barrier Function In Vivo by Biotin

    PubMed Central

    Ding, Lei; Zhang, Yuguo; Tatum, Rodney; Chen, Yan-Hua

    2011-01-01

    Tight junctions (TJs) are the most apical component of the junctional complexes in mammalian epithelial cells and form selective paracellular barriers restricting the passage of solutes and ions across the epithelial sheets. Claudins, a TJ integral membrane protein family, play a critical role in regulating paracellular barrier permeability. In the in vitro cell culture system, transepithelial electrical resistance (TER) measurement and the flux of radioisotope or fluorescent labeled molecules with different sizes have been widely used to determine the TJ barrier function. In the in vivo system, the tracer molecule Sulfo-NHS-Biotin was initially used in Xenopus embryos system and subsequently was successfully applied to a number of animal tissues in situ and in different organisms under the experimental conditions to examine the functional integrity of TJs by several laboratories. In this chapter, we will describe the detailed procedures of applying biotin as a paracellular tracer molecule to different in vivo systems to assay TJ barrier function. PMID:21717351

  8. In vivo and in vitro analysis of the human tissue-type plasminogen activator gene promoter in neuroblastomal cell lines: evidence for a functional upstream kappaB element.

    PubMed

    Lux, W; Klobeck, H-G; Daniel, P B; Costa, M; Medcalf, R L; Schleuning, W-D

    2005-05-01

    Besides its well-established role in wound healing and fibrinolysis, tissue-type plasminogen activator (t-PA) has been shown to contribute to cognitive processes and memory formation within the central nervous system, and to promote glutamate receptor-mediated excitotoxicity. The t-PA gene is expressed and regulated in neuronal cells but the regulatory transcriptional processes directing this expression are still poorly characterized. We have used DNase I-hypersensitivity mapping and in vivo foot printing to identify putative regulatory elements and transcription factor binding sites in two human neuroblastomal (KELLY and SK-N-SH) and one human glioblastomal (SNB-19) cell lines. Hypersensitive sites were found in the proximal promoter region of all cell lines, and within the first exon for KELLY and SNB-19 cells. Mapping of methylation-protected residues in vivo detected a cluster of protected residues corresponding to a cAMP response element (CRE) and Sp1 sites in the proximal promoter previously shown to be essential for basal expression in other cell types. Protected residues were also found at other sites, notably a kappaB element at position bp -3081 to -3072 that was partly protected in KELLY and SNB-19 cells. Analysis of transfected reporter constructs in KELLY and SNB-19 cells confirmed that this particular element is functionally significant in the transactivation of the t-PA promoter in both cell types. This study defines, by in vivo and in vitro methods, a previously undescribed kappaB site in the t-PA gene promoter that influences t-PA expression in neuronal cells. PMID:15869598

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

    PubMed

    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

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

    PubMed

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

    2014-10-01

    Amphioxus is a promising new animal model for developmental biology. To develop molecular tools for this model, we characterized the promoter region of a cytoplasmic β-actin gene (Bb-actin-6-2) from the Chinese amphioxus Branchiostoma belcheri. In situ hybridization and real time-quantitative PCR analyses showed that this gene is expressed in many tissues throughout embryonic development. Cloning of cDNA revealed two isoforms with distinct transcription start sites. Isoform #1 exhibits a similar exon/intron and regulatory element organization to that of vertebrate β-actin, whereas isoform #2 lacks the first exon of isoform #1 and recruits its first intron as a promoter. The activities of upstream promoter regions in the two isoforms were examined using the lacZ reporter system in amphioxus embryos. The proximal promoter of isoform #1 drove reporter gene expression broadly in 58.6 % of injected embryos. That of isoform #2 exhibited much higher activity (91.5 %) than that of isoform #1 or the human EF-1-α gene (38.2 %). We determined the minimal promoter regions of the two isoforms via functional analysis. These two regions, alone or inserted a random DNA fragment upstream, had no detectable activity, but when an upstream enhancer was inserted, the promoters directed reporter gene expression in 61.0 and 93.8 %, respectively, of injected embryos in a tissue-specific manner. Our study not only provides insight into the regulatory mechanism underlying amphioxus Bb-actin-6-2 gene expression, but also identifies two sets of efficient proximal and minimal promoters. These promoters could be used to construct gene expression vectors for transgenic studies using amphioxus as a model. PMID:25078982

  11. 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

  12. In Vivo Gait Analysis During Bone Transport.

    PubMed

    Mora-Macías, J; Reina-Romo, E; Morgaz, J; Domínguez, J

    2015-09-01

    The load bearing characteristics of the intervened limb over time in vivo are important to know in distraction osteogenesis and bone healing for the characterization of the bone maturation process. Gait analyses were performed for a group of sheep in which bone transport was carried out. The ground reaction force was measured by means of a force platform, and the gait parameters (i.e., the peak, the mean vertical ground reaction force and the impulse) were calculated during the stance phase for each limb. The results showed that these gait parameters decreased in the intervened limb and interestingly increased in the other limbs due to the implantation of the fixator. Additionally, during the process, the gait parameters exponentially approached the values for healthy animals. Corresponding radiographies showed an increasing level of ossification in the callus. This study shows, as a preliminary approach to be confirmed with more experiments, that gait analysis could be used as an alternative method to control distraction osteogenesis or bone healing. For example, these analyses could determine the appropriate time to remove the fixator. Furthermore, gait analysis has advantages over other methods because it provides quantitative data and does not require instrumented fixators. PMID:25650097

  13. Free-radical probes for functional in vivo EPR imaging

    NASA Astrophysics Data System (ADS)

    Subramanian, S.; Krishna, M. C.

    2007-02-01

    Electron paramagnetic resonance imaging (EPRI) is one of the recent functional imaging modalities that can provide valuable in vivo physiological information on its own merit and aids as a complimentary imaging technique to MRI and PET of tissues especially with respect to in vivo pO II (oxygen partial pressure), redox status and pharmacology. EPR imaging mainly deals with the measurement of distribution and in vivo dynamics and redox changes using special nontoxic paramagnetic spin probes that can be infused into the object of investigation. These spin probes should be characterized by simple EPR spectra, preferably with narrow EPR lines. The line width should be reversibly sensitive to the concentration of in vivo pO II with a linear dependence. Several non-toxic paramagnetic probes, some particulate and insoluble and others water-soluble and infusible (by intravenous or intramuscular injection) have been developed which can be effectively used to quantitatively assess tissue redox status, and tumor hypoxia. Quantitative assessment of the redox status of tissue in vivo is important in investigating oxidative stress, and that of tissue pO II is very important in radiation oncology. Other areas in which EPR imaging and oxymetry may help are in the investigation of tumorangiogenesis, wound healing, oxygenation of tumor tissue by the ingestion of oxygen-rich gases, etc. The correct choice of the spin probe will depend on the modality of measurement (whether by CW or time-domain EPR imaging) and the particular physiology interrogated. Examples of the available spin probes and some EPR imaging applications employing them are presented.

  14. Preparation and functional characterization of human vascular endothelial growth factor-melittin fusion protein with analysis of the antitumor activity in vitro and in vivo.

    PubMed

    Wang, Dingding; Hu, Lili; Su, Manman; Wang, Ju; Xu, Tianmin

    2015-09-01

    Vascular endothelial growth factor and its tyrosine kinase receptors have been identified as key mediators of the regulation of pathologic blood vessel growth and maintenance in the promotion of angiogenesis and tumor growth. Therefore, an alternative approach to destroying tumor endothelium would be to make this tissue particularly sensitive to VEGF-mediated drug delivery. To verify this hypothesis, we generated a protein containing VEGF165 fused to melittin. Melittin is a small linear peptide composed of 26 amino acid residues that can exert toxic or inhibitory effects on many types of tumor cells. This protein is a cytolytic peptide that attacks lipid membranes, leading to significant toxicity. In the present study, the Pichia pastoris expression system was used to express the fusion protein. Under optimal conditions, stable VEGF165-melittin production was achieved using a series of purification steps. The activity of VEGF165-melittin fusion protein was compared with melittin for its ability to suppress the growth of tumor cell line in vitro. The fusion toxin selectively inhibited growth of human hepatocellular carcinoma HepG-2 cell line with high expression of VEGFR-2. We found that sensitivity of VEGFR-2 transfected 293 cells to VEGF165-melittin enhanced as the cellular VEGFR-2 density increased. In an in vivo initial experiment, the fusion protein inhibited tumor growth in xenografts assays. Furthermore, successful expression and characterization of the fusion protein demonstrated its efficacy for use as a novel treatment strategy for cancer. PMID:26166416

  15. Recent molecular approaches to understanding astrocyte function in vivo

    PubMed Central

    Davila, David; Thibault, Karine; Fiacco, Todd A.; Agulhon, Cendra

    2013-01-01

    Astrocytes are a predominant glial cell type in the nervous systems, and are becoming recognized as important mediators of normal brain function as well as neurodevelopmental, neurological, and neurodegenerative brain diseases. Although numerous potential mechanisms have been proposed to explain the role of astrocytes in the normal and diseased brain, research into the physiological relevance of these mechanisms in vivo is just beginning. In this review, we will summarize recent developments in innovative and powerful molecular approaches, including knockout mouse models, transgenic mouse models, and astrocyte-targeted gene transfer/expression, which have led to advances in understanding astrocyte biology in vivo that were heretofore inaccessible to experimentation. We will examine the recently improved understanding of the roles of astrocytes – with an emphasis on astrocyte signaling – in the context of both the healthy and diseased brain, discuss areas where the role of astrocytes remains debated, and suggest new research directions. PMID:24399932

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

    PubMed

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

    2015-07-01

    Intravital imaging using multiphoton microscopy (MPM) has become an increasingly popular and widely used experimental technique in kidney research over the past few years. MPM allows deep optical sectioning of the intact, living kidney tissue with submicron resolution, which is unparalleled among intravital imaging approaches. MPM has solved a long-standing critical technical barrier in renal research to study several complex and inaccessible cell types and anatomical structures in vivo in their native environment. Comprehensive and quantitative kidney structure and function MPM studies helped our better understanding of the cellular and molecular mechanisms of the healthy and diseased kidney. This review summarizes recent in vivo MPM studies with a focus on the glomerulus and the filtration barrier, although select, glomerulus-related renal vascular and tubular functions are also mentioned. The latest applications of serial MPM of the same glomerulus in vivo, in the intact kidney over several days, during the progression of glomerular disease are discussed. This visual approach, in combination with genetically encoded fluorescent markers of cell lineage, has helped track the fate and function (e.g., cell calcium changes) of single podocytes during the development of glomerular pathologies, and provided visual proof for the highly dynamic, rather than static, nature of the glomerular environment. Future intravital imaging applications have the promise to further push the limits of optical microscopy, and to advance our understanding of the mechanisms of kidney injury. Also, MPM will help to study new mechanisms of tissue repair and regeneration, a cutting-edge area of kidney research. PMID:25738253

  17. Algal photoreceptors: in vivo functions and potential applications.

    PubMed

    Kianianmomeni, Arash; Hallmann, Armin

    2014-01-01

    Many algae, particularly microalgae, possess a sophisticated light-sensing system including photoreceptors and light-modulated signaling pathways to sense environmental information and secure the survival in a rapidly changing environment. Over the last couple of years, the multifaceted world of algal photobiology has enriched our understanding of the light absorption mechanisms and in vivo function of photoreceptors. Moreover, specific light-sensitive modules have already paved the way for the development of optogenetic tools to generate light switches for precise and spatial control of signaling pathways in individual cells and even in complex biological systems. PMID:24081482

  18. 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

  19. HIV control in vivo: Dynamical analysis

    NASA Astrophysics Data System (ADS)

    Gumel, A. B.; Moghadas, S. M.

    2004-10-01

    A deterministic model for the immunological and therapeutic control of human immunodeficiency virus (HIV) in vivo is studied qualitatively. In addition to analyzing the local stability of the equilibria, the global stability of the infection-free equilibrium is established. The optimal efficacy level of anti-retroviral therapy needed to eradicate HIV from the body of an HIV-infected individual is obtained.

  20. In vivo minimally invasive interstitial multi-functional microendoscopy

    PubMed Central

    Shahmoon, Asaf; Aharon, Shiran; Kruchik, Oded; Hohmann, Martin; Slovin, Hamutal; Douplik, Alexandre; Zalevsky, Zeev

    2013-01-01

    Developing minimally invasive methodologies for imaging of internal organs is an emerging field in the biomedical examination research. This paper introduces a new multi-functional microendoscope device capable of imaging of internal organs with a minimal invasive intervention. In addition, the developed microendoscope can also be employed as a monitoring device for measuring local hemoglobin concentration in blood stream when administrated into a blood artery. The microendoscope device has a total external diameter of only 200 μm and can provide high imaging resolution capability of more than 5,000 pixels. The device can detect features with a spatial resolution of less than 1 μm. The microendoscope has been tested both in-vitro as well as in-vivo in rats presenting a promising and powerful tool as a high resolution and minimally invasive imaging facility suitable for previously unreachable clinical modalities. PMID:23712369

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

    NASA Astrophysics Data System (ADS)

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

    1999-07-01

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

  2. Intracranial nonthermal irreversible electroporation: in vivo analysis.

    PubMed

    Garcia, Paulo A; Rossmeisl, John H; Neal, Robert E; Ellis, Thomas L; Olson, John D; Henao-Guerrero, Natalia; Robertson, John; Davalos, Rafael V

    2010-07-01

    Nonthermal irreversible electroporation (NTIRE) is a new minimally invasive technique to treat cancer. It is unique because of its nonthermal mechanism of tumor ablation. Intracranial NTIRE procedures involve placing electrodes into the targeted area of the brain and delivering a series of short but intense electric pulses. The electric pulses induce irreversible structural changes in cell membranes, leading to cell death. We correlated NTIRE lesion volumes in normal brain tissue with electric field distributions from comprehensive numerical models. The electrical conductivity of brain tissue was extrapolated from the measured in vivo data and the numerical models. Using this, we present results on the electric field threshold necessary to induce NTIRE lesions (495-510 V/cm) in canine brain tissue using 90 50-mus pulses at 4 Hz. Furthermore, this preliminary study provides some of the necessary numerical tools for using NTIRE as a brain cancer treatment. We also computed the electrical conductivity of brain tissue from the in vivo data (0.12-0.30 S/m) and provide guidelines for treatment planning and execution. Knowledge of the dynamic electrical conductivity of the tissue and electric field that correlates to lesion volume is crucial to ensure predictable complete NTIRE treatment while minimizing damage to surrounding healthy tissue. PMID:20668843

  3. 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

  4. 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.

  5. Characteristics and Functional Roles of Opioids Originally Present in Vivo.

    PubMed

    Ozaki, Masanobu

    2016-01-01

    The characteristics and functional roles of opioids originally present in vivo (endogenous opioids) in guinea-pig ileum were investigated. The release of endogenous opioids was determined by the inhibitory twitch response evoked by 0.1 Hz stimulation after 10 Hz stimulation (post-tetanic twitch inhibition). The effects of peptidase inhibitors increased the post-tetanic twitch inhibition, prevented by β-funaltrexamine and nor-binaltorphimine, which are selective μ- and κ-opioid receptor subtype antagonists, respectively. Dopamine receptor antagonists (haloperidol, sultopride and domperidone) increased the post-tetanic twitch inhibition. These results suggest that dopamine receptors are involved in modulation of the ileal opioid system, so as to diminish endogenous opioid release by tetanic stimulation, and dopamine antagonists increase the opioid action, that might depend more on the increased release of endogenous opioids. The post-tetanic twitch inhibition was inhibited by adrenalectomy, and showed the supersensitivity of the opioid receptors, resulting from a decrease of endogenous opioids by adrenalectomy. These findings suggest that the increase in morphine-analgesia by adrenalectomy was due to this process. In the presence of naloxone, an opioid antagonist, an increase in basal tension after tetanic stimulation (10 Hz stimulation) (post-tetanic contraction) was observed, and was blocked by spantide, a substance P antagonist, and indomethacin, a prostaglandins-biosynthesis inhibitor. This contraction increased with morphine or peptidase inhibitor exposure, depending on the length of time the ileum was exposed to the morphine or peptidase inhibitor. Post-tetanic contraction might be a useful indicator of the formation of physical dependence to morphine or endogenous opioids in the ileum. PMID:27040344

  6. Fetal in vivo continuous cardiovascular function during chronic hypoxia.

    PubMed

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

    2016-03-01

    Although the fetal cardiovascular defence to acute hypoxia and the physiology underlying it have been established for decades, how the fetal cardiovascular system responds to chronic hypoxia has been comparatively understudied. We designed and created isobaric hypoxic chambers able to maintain pregnant sheep for prolonged periods of gestation under controlled significant (10% O2) hypoxia, yielding fetal mean P(aO2) levels (11.5 ± 0.6 mmHg) similar to those measured in human fetuses of hypoxic pregnancy. We also created a wireless data acquisition system able to record fetal blood flow signals in addition to fetal blood pressure and heart rate from free moving ewes as the hypoxic pregnancy is developing. We determined in vivo longitudinal changes in fetal cardiovascular function including parallel measurement of fetal carotid and femoral blood flow and oxygen and glucose delivery during the last third of gestation. The ratio of oxygen (from 2.7 ± 0.2 to 3.8 ± 0.8; P < 0.05) and of glucose (from 2.3 ± 0.1 to 3.3 ± 0.6; P < 0.05) delivery to the fetal carotid, relative to the fetal femoral circulation, increased during and shortly after the period of chronic hypoxia. In contrast, oxygen and glucose delivery remained unchanged from baseline in normoxic fetuses. Fetal plasma urate concentration increased significantly during chronic hypoxia but not during normoxia (Δ: 4.8 ± 1.6 vs. 0.5 ± 1.4 μmol l(-1), P<0.05). The data support the hypotheses tested and show persisting redistribution of substrate delivery away from peripheral and towards essential circulations in the chronically hypoxic fetus, associated with increases in xanthine oxidase-derived reactive oxygen species. PMID:26926316

  7. Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain.

    PubMed

    Wang, Xueding; Pang, Yongjiang; Ku, Geng; Xie, Xueyi; Stoica, George; Wang, Lihong V

    2003-07-01

    Imaging techniques based on optical contrast analysis can be used to visualize dynamic and functional properties of the nervous system via optical signals resulting from changes in blood volume, oxygen consumption and cellular swelling associated with brain physiology and pathology. Here we report in vivo noninvasive transdermal and transcranial imaging of the structure and function of rat brains by means of laser-induced photoacoustic tomography (PAT). The advantage of PAT over pure optical imaging is that it retains intrinsic optical contrast characteristics while taking advantage of the diffraction-limited high spatial resolution of ultrasound. We accurately mapped rat brain structures, with and without lesions, and functional cerebral hemodynamic changes in cortical blood vessels around the whisker-barrel cortex in response to whisker stimulation. We also imaged hyperoxia- and hypoxia-induced cerebral hemodynamic changes. This neuroimaging modality holds promise for applications in neurophysiology, neuropathology and neurotherapy. PMID:12808463

  8. Analysis of the structure of human telomerase RNA in vivo

    PubMed Central

    Antal, Mária; Boros, Éva; Solymosy, Ferenc; Kiss, Tamás

    2002-01-01

    Telomerase is a ribonucleoprotein reverse transcriptase that synthesises telomeric DNA. The RNA component of telomerase acts as a template for telomere synthesis and binds the reverse transcriptase. In this study, we have performed in vivo and in vitro structural analyses of human telomerase RNA (hTR). In vivo mapping experiments showed that the 5′-terminal template domain of hTR folds into a long hairpin structure, in which the template sequence occupies a readily accessible position. Intriguingly, neither in vivo nor in vitro mapping of hTR confirmed formation of a stable ‘pseudoknot’ helix, suggesting that this functionally essential long range interaction is formed only temporarily. In vitro control mappings demonstrated that the 5′-terminal template domain of hTR cannot fold correctly in the absence of cellular protein factors. The 3′-terminal domain of hTR, both in vivo and in vitro, folds into the previously predicted box H/ACA snoRNA-like ‘hairpin–hinge–hairpin–tail’ structure. Finally, comparison of the in vivo and in vitro modification patterns of hTR revealed several regions that might be directly involved in binding of telomerase reverse transcriptase or other telomerase proteins. PMID:11842102

  9. 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

  10. In vivo analysis of recycling endosomes in Caenorhabditis elegans.

    PubMed

    Shi, Anbing; Grant, Barth D

    2015-01-01

    The microscopic nematode Caenorhabditis elegans (C. elegans) serves as an excellent animal model for studying membrane traffic. This is due in part to its highly advanced genetics and genomics, and a transparent body that allows the visualization of fluorescently tagged molecules in the physiologically relevant context of the intact organism. Notably, C. elegans oocytes, coelomocytes, and intestinal epithelia have been established as facile cellular models to explore nonpolarized and polarized cell membrane trafficking mechanisms. In this chapter, we describe in vivo C. elegans assays, utilizing fluorescent dyes or proteins, to examine the molecular mechanisms that control endocytosis and endocytic recycling. Tissue-specific, steady-state imaging and associated quantitative analysis allow the identification and interpretation of subcellular events in the intact animal. To better understand the kinetic characteristics of recycling tubules that mediate membrane protein recycling, we describe recently developed dynamic-imaging assays in intestinal epithelial cells. Such methods bring new clarity to the system, helping to elucidate the functional roles of recycling mediators. PMID:26360035

  11. Quantitative analysis of in vivo cell proliferation.

    PubMed

    Cameron, Heather A

    2006-11-01

    Injection and immunohistochemical detection of 5-bromo-2'-deoxyuridine (BrdU) has become the standard method for studying the birth and survival of neurons, glia, and other cell types in the nervous system. BrdU, a thymidine analog, becomes stably incorporated into DNA during the S-phase of mitosis. Because DNA containing BrdU can be specifically recognized by antibodies, this method allows dividing cells to be marked at any given time and then identified at time points from a few minutes to several years later. BrdU immunohistochemistry is suitable for cell counting to examine the regulation of cell proliferation and cell fate. It can be combined with labeling by other antibodies, allowing confocal analysis of cell phenotype or expression of other proteins. The potential for nonspecific labeling and toxicity are discussed. Although BrdU immunohistochemistry has almost completely replaced tritiated thymidine autoradiography for labeling dividing cells, this method and situations in which it is still useful are also described. PMID:18428635

  12. 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…

  13. In vivo TRPC functions in the cardiopulmonary vasculature.

    PubMed

    Dietrich, Alexander; Kalwa, Hermann; Fuchs, Beate; Grimminger, Friedrich; Weissmann, Norbert; Gudermann, Thomas

    2007-08-01

    Cardiovascular diseases are the leading cause of death in the industrialized countries. The cardiovascular system includes the systemic blood circulation, the heart and the pulmonary circulation providing sufficient blood flow and oxygen to peripheral tissues and organs according to their metabolic demand. This review focuses on three major cell types of the cardiovascular system: myocytes of the heart as well as smooth muscle cells and endothelial cells from the systemic and pulmonary circulation. Ion channels initiate and regulate contraction in all three cell types, and the identification of their genes has significantly improved our knowledge of signal transduction pathways in these cells. Among the ion channels expressed in smooth muscle cells, cation channels of the TRPC family allow for the entry of Na(+) and Ca(2+). Physiological functions of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 in the cardiovascular system, dissected by down-regulating channel activity in isolated tissues or by the analysis of gene-deficient mouse models, are reviewed. Possible functional roles and physiological regulation of TRPCs as homomeric or heteromeric channels in these cell types are discussed. Moreover, TRP channels may also be responsible for pathophysiological processes of the cardiovascular system like hypertension as well as cardiac hypertrophy and increased endothelial permeability. PMID:17433435

  14. 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.

  15. Blood-Induced Interference of Glucose Sensor Function in Vitro: Implications for in Vivo Sensor Function

    PubMed Central

    Klueh, Ulrike; Liu, Zenghe; Ouyang, Tianmei; Cho, Brian; Feldman, Ben; Henning, Timothy P.; Kreutzer, Don

    2007-01-01

    Background Although tissue hemorrhages, with resulting blood clots, are associated with glucose sensor implantation, virtually nothing known is about the impact of red blood cells and red blood cell clots on sensor function in vitro or in vivo. In these studies, we tested the hypothesis that blood can directly interfere with glucose sensor function in vitro. Methods To test this hypothesis, heparinized human whole blood (HWB) and nonheparinized human whole blood (WB) were obtained from normal individuals. Aliquots of HWB and WB samples were also fractionated into plasma, serum, and total leukocyte (TL) components. Resulting HWB, WB, and WB components were incubated in vitro with an amperometric glucose sensor for 24 hours at 37°C. During incubation, blood glucose levels were determined periodically using a glucose monitor, and glucose sensor function (GSF) was monitored continuously as nanoampere output. Results Heparinized human whole blood had no significant effect on GSF in vitro, nor did TL, serum, or plasmaderived clots from WB. Sensors incubated with WB displayed a rapid signal loss associated with clot formation at 37°C. The half-life was 0.8 ± 0.2 hours (n = 16) for sensors incubated with WB compared to 3.2 ± 0.5 (n = 12) for sensors incubated with HWB with a blood glucose level of approximately 100 mg/dl. Conclusions These studies demonstrated that human whole blood interfered with GSF in vitro. These studies further demonstrated that this interference was related to blood clot formation, as HWB, serum, plasma-derived clots, or TL did not interfere with GSF in vitro in the same way that WB did. These in vitro studies supported the concept that the formation of blood clots at sites of glucose sensor implantation could have a negative impact on GSF in vivo. PMID:19885155

  16. In vivo imaging of functional disruption, recovery and alteration in rat olfactory circuitry after lesion.

    PubMed

    Cross, Donna J; Flexman, Jennifer A; Anzai, Yoshimi; Morrow, Thomas J; Maravilla, Kenneth R; Minoshima, Satoshi

    2006-09-01

    Compensatory changes following disruption of neuronal circuitry have been indicated by previous imaging studies of stroke and other brain injury, but evidence of the pathways involved in such dynamic changes has not been shown in vivo. We imaged rats before and after lesion-induced disruption of the lateral olfactory tract to investigate the subsequent recovery and/or reorganization of functional neuronal circuitry. Serial magnetic resonance imaging was performed following intranasal administration of a paramagnetic track tracer Mn(2+). Images were analyzed using statistical mapping techniques in the stereotactic coordinate system. At 1 week post-lesion, Mn(2+) transport caudal to lesion was reduced as expected, and more importantly, increased transport through the anterior commissure was seen. At 4 weeks post-lesion, there was recovery of transport caudal to lesion, and increased transport through the anterior commissure extended to the contralateral olfactory cortex. Correlation analysis of regional Mn(2+) transport indicated that contralateral enhancement was not simply due to septal window spillover. This study demonstrates for the first time in vivo evidence of compensatory changes in functional neuronal activity to a contralateral pathway through the commissure following brain injury. PMID:16859928

  17. Functional Genetic Targeting of Embryonic Kidney Progenitor Cells Ex Vivo

    PubMed Central

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

    2015-01-01

    The embryonic mammalian metanephric mesenchyme (MM) is a unique tissue because it is competent to generate the nephrons in response to Wnt signaling. An ex vivo culture in which the MM is separated from the ureteric bud (UB), the natural inducer, can be used as a classic tubule induction model for studying nephrogenesis. However, technological restrictions currently prevent using this model to study the molecular genetic details before or during tubule induction. Using nephron segment-specific markers, we now show that tubule induction in the MM ex vivo also leads to the assembly of highly segmented nephrons. This induction capacity was reconstituted when MM tissue was dissociated into a cell suspension and then reaggregated (drMM) in the presence of human recombinant bone morphogenetic protein 7/human recombinant fibroblast growth factor 2 for 24 hours before induction. Growth factor–treated drMM also recovered the capacity for organogenesis when recombined with the UB. Cell tracking and time-lapse imaging of chimeric drMM cultures indicated that the nephron is not derived from a single progenitor cell. Furthermore, viral vector-mediated transduction of green fluorescent protein was much more efficient in dissociated MM cells than in intact mesenchyme, and the nephrogenic competence of transduced drMM progenitor cells was preserved. Moreover, drMM cells transduced with viral vectors mediating Lhx1 knockdown were excluded from the nephric tubules, whereas cells transduced with control vectors were incorporated. In summary, these techniques allow reproducible cellular and molecular examinations of the mechanisms behind nephrogenesis and kidney organogenesis in an ex vivo organ culture/organoid setting. PMID:25201883

  18. In vivo characterization of regenerative peripheral nerve interface function

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

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

    PubMed

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

    2016-01-01

    Acupuncture treatment is amazing but controversial. Up to now, the mechanism of treating diseases by acupuncture and moxibustion is still unclear, especially the occurrence of the molecular events in local acupoints. Herein, we report an extremely stable microsensor by modifying carbon nanotube (CNT) to the tip surface of acupuncture needle and applying this CNT-modified acupuncture needle for real time monitoring of serotonin (5-HT) in vivo. To stabilize CNT modification on the needle tip surface, poly(3,4-ethylenedioxythiophene)(PEDOT) was employed as glue water to stick CNT on the needle. The detection limit of the CNT-modified needle was found to be approximately 50 nM and 78 nM in the PBS and the cell medium, respectively. In addition, the needle showed good selectivity to some inflammatory mediators and some electroactive molecules. For the first time, the CNT-modified needle could be directly probed into rat body for real time monitoring of 5-HT in vivo, showing a great potential for better understanding the mechanism of acupuncture treatment. PMID:27301303

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

    Acupuncture treatment is amazing but controversial. Up to now, the mechanism of treating diseases by acupuncture and moxibustion is still unclear, especially the occurrence of the molecular events in local acupoints. Herein, we report an extremely stable microsensor by modifying carbon nanotube (CNT) to the tip surface of acupuncture needle and applying this CNT-modified acupuncture needle for real time monitoring of serotonin (5-HT) in vivo. To stabilize CNT modification on the needle tip surface, poly(3,4-ethylenedioxythiophene)(PEDOT) was employed as glue water to stick CNT on the needle. The detection limit of the CNT-modified needle was found to be approximately 50 nM and 78 nM in the PBS and the cell medium, respectively. In addition, the needle showed good selectivity to some inflammatory mediators and some electroactive molecules. For the first time, the CNT-modified needle could be directly probed into rat body for real time monitoring of 5-HT in vivo, showing a great potential for better understanding the mechanism of acupuncture treatment. PMID:27301303

  2. 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

  3. Statistical mapping of functional olfactory connections of the rat brain in vivo.

    PubMed

    Cross, Donna J; Minoshima, Satoshi; Anzai, Yoshimi; Flexman, Jennifer A; Keogh, Bartholomew P; Kim, Yongmin; Maravilla, Kenneth R

    2004-12-01

    The olfactory pathway is a unique route into the brain. To better characterize this system in vivo, rat olfactory functional connections were mapped using magnetic resonance (MR) imaging and manganese ion (Mn2+) as a transport-mediated tracer combined with newly developed statistical brain image analysis. Six rats underwent imaging on a 1.5-T MR scanner at pre-administration, and 6, 12, 24, 36, 48, and 72 h and 5.5, 7.5, 10.5, and 13.5 days post-administration of manganese chloride (MnCl2) into the right nasal cavity. Images were coregistered, pixel-intensity normalized, and stereotactically transformed to the Paxinos and Watson rat brain atlas, then averaged across subjects using automated image analysis software (NEUROSTAT). Images at each time point were compared to pre-administration using a one-sample t statistic on a pixel-by-pixel basis in 3-D and converted to Z statistic maps. Statistical mapping and group averaging improved signal to noise ratios and signal detection sensitivity. Significant transport of Mn2+ was observed in olfactory structures ipsilateral to site of Mn2+ administration including the bulb, lateral olfactory tract (lo) by 12 h and in the tubercle, piriform cortex, ventral pallidum, amygdala, and in smaller structures such as the anterior commissure after 24 h post-administration. MR imaging with group-wise statistical analysis clearly demonstrated bilateral transsynaptic Mn2+ transport to secondary and tertiary neurons of the olfactory system. The method permits in vivo investigations of functional neuronal connections within the brain. PMID:15589097

  4. HLA-B27/microbial mimicry: an in vivo analysis.

    PubMed Central

    Kapasi, K; Chui, B; Inman, R D

    1992-01-01

    The association between three major spondyloarthritic diseases, ankylosing spondylitis, Reiter's syndrome, and reactive arthritis, and the major histocompatibility complex (MHC) class 1 antigen HLA-B27 is well documented. The hypothesis of cross-reactivity between HLA-B27 and the antecedent infection-causing Gram-negative pathogens such as Salmonella, Shigella and Yersinia has been suggested by in vitro studies employing monoclonal antibodies. We have examined the possibility of such cross-reactivity in vivo using various rabbit immune sera and patient sera as the source of cross-reacting antibody. Mouse L cells were transfected with HLA-A3 or HLA-B27 and used as a source of antigen. Western blot analysis employing denatured antigen, FACS analysis employing native antigen and immunoprecipitation studies were undertaken to detect cross-reacting antibodies generated in vivo to HLA-B27 antigen. Antibodies generated in vivo by infection in patients or immunization in animals against arthritogenic bacteria did not demonstrate any cross-reactivity with HLA-B27 by any of the methods used. As defined by the humoral immune response, molecular mimicry appears unlikely to explain the role of B27 in the pathogenesis of reactive arthritis. Images Figure 2 Figure 3 Figure 6 PMID:1478690

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

    PubMed Central

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

    2012-01-01

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

  6. 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,…

  7. Function Analysis and Decomposistion using Function Analysis Systems Technique

    SciTech Connect

    J. R. Wixson

    1999-06-01

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

  8. Function Analysis and Decomposistion using Function Analysis Systems Technique

    SciTech Connect

    Wixson, James Robert

    1999-06-01

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

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

    Cancer.gov

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

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

    SciTech Connect

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

    1984-02-01

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

  11. In vivo analysis of wound healing by optical methods

    PubMed Central

    Alborova, Alena; Lademann, Jürgen; Kramer, Axel; Richter, Heike; Patzelt, Alexa; Sterry, Wolfram; Koch, Stefan

    2008-01-01

    The analysis of wound healing is important for the therapy control and for the development of drugs stimulating the healing process. Wounds cause damage to the skin barrier. A damaged stratum corneum leads to an increased water loss through the skin barrier. The standard measuring procedure for characterization of wound healing is the measurement of transepidermal water loss (TEWL). The disadvantage of this method is that it can be easily disturbed by the perspiration of the volunteers and by topically applied substances, for instance wound healing creams. In the study presented, in vivo laser scanning microscopy and optical coherent tomography were compared concerning the application for their analysis of wound healing processes. The laser scanning microscopy allows the analysis of the healing process on a cellular level. The course of wound healing determined by laser scanning microscopy was correlated with numerical values, allowing the numerical characterization of the wound healing process. PMID:20204112

  12. GABAA receptor modulating steroid antagonists (GAMSA) are functional in vivo.

    PubMed

    Johansson, Maja; Strömberg, Jessica; Ragagnin, Gianna; Doverskog, Magnus; Bäckström, Torbjörn

    2016-06-01

    GABAA receptor modulating steroid antagonists (GAMSA) selectively inhibit neurosteroid-mediated enhancement of GABA-evoked currents at the GABAA receptor. 3α-hydroxy-neurosteroids, notably allopregnanolone and tetrahydrodeoxycorticosterone (THDOC), potentiate GABAA receptor-mediated currents. On the contrary, various 3β-hydroxy-steroids antagonize this positive neurosteroid-mediated modulation. Importantly, GAMSAs are specific antagonists of the positive neurosteroid-modulation of the receptor and do not inhibit GABA-evoked currents. Allopregnanolone and THDOC have both negative and positive actions. Allopregnanolone can impair encoding/consolidation and retrieval of memories. Chronic administration of a physiological allopregnanolone concentration reduces cognition in mice models of Alzheimer's disease. In humans an allopregnanolone challenge impairs episodic memory and in hepatic encephalopathy cognitive deficits are accompanied by increased brain ammonia and allopregnanolone. Hippocampal slices react in vitro to ammonia by allopregnanolone synthesis in CA1 neurons, which blocks long-term potentiation (LTP). Thus, allopregnanolone may impair learning and memory by interfering with hippocampal LTP. Contrary, pharmacological treatment with allopregnanolone can promote neurogenesis and positively influence learning and memory of trace eye-blink conditioning in mice. In rat the GAMSA UC1011 inhibits an allopregnanolone-induced learning impairment and the GAMSA GR3027 restores learning and motor coordination in rats with hepatic encephalopathy. In addition, the GAMSA isoallopregnanolone antagonizes allopregnanolone-induced anesthesia in rats, and in humans it antagonizes allopregnanolone-induced sedation and reductions in saccadic eye velocity. 17PA is also an effective GAMSA in vivo, as it antagonizes allopregnanolone-induced anesthesia and spinal analgesia in rats. In vitro the allopregnanolone/THDOC-increased GABA-mediated GABAA receptor activity is antagonized

  13. 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...

  14. 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.

  15. Specific in vivo knockdown of protein function by intrabodies

    PubMed Central

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

    2015-01-01

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

  16. In vivo analysis of burns in a mouse model using spectroscopic optical coherence tomography.

    PubMed

    Maher, Jason R; Jaedicke, Volker; Medina, Manuel; Levinson, Howard; Selim, Maria Angelica; Brown, William J; Wax, Adam

    2014-10-01

    Spectroscopic analysis of biological tissues can provide insight into changes in structure and function due to disease or injury. Depth-resolved spectroscopic measurements can be implemented for tissue imaging using optical coherence tomography (OCT). Here, spectroscopic OCT is applied to in vivo measurement of burn injury in a mouse model. Data processing and analysis methods are compared for their accuracy. Overall accuracy in classifying burned tissue was found to be as high as 91%, producing an area under the curve of a receiver operating characteristic curve of 0.97. The origins of the spectral changes are identified by correlation with histopathology. PMID:25360936

  17. A guide to human in vivo microcirculatory flow image analysis.

    PubMed

    Massey, Michael J; Shapiro, Nathan I

    2016-01-01

    Various noninvasive microscopic camera technologies have been used to visualize the sublingual microcirculation in patients. We describe a comprehensive approach to bedside in vivo sublingual microcirculation video image capture and analysis techniques in the human clinical setting. We present a user perspective and guide suitable for clinical researchers and developers interested in the capture and analysis of sublingual microcirculatory flow videos. We review basic differences in the cameras, optics, light sources, operation, and digital image capture. We describe common techniques for image acquisition and discuss aspects of video data management, including data transfer, metadata, and database design and utilization to facilitate the image analysis pipeline. We outline image analysis techniques and reporting including video preprocessing and image quality evaluation. Finally, we propose a framework for future directions in the field of microcirculatory flow videomicroscopy acquisition and analysis. Although automated scoring systems have not been sufficiently robust for widespread clinical or research use to date, we discuss promising innovations that are driving new development. PMID:26861691

  18. 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.

  19. Genome-scale analysis of in vivo spatiotemporal promoter activity in Caenorhabditis elegans.

    PubMed

    Dupuy, Denis; Bertin, Nicolas; Hidalgo, César A; Venkatesan, Kavitha; Tu, Domena; Lee, David; Rosenberg, Jennifer; Svrzikapa, Nenad; Blanc, Aurélie; Carnec, Alain; Carvunis, Anne-Ruxandra; Pulak, Rock; Shingles, Jane; Reece-Hoyes, John; Hunt-Newbury, Rebecca; Viveiros, Ryan; Mohler, William A; Tasan, Murat; Roth, Frederick P; Le Peuch, Christian; Hope, Ian A; Johnsen, Robert; Moerman, Donald G; Barabási, Albert-László; Baillie, David; Vidal, Marc

    2007-06-01

    Differential regulation of gene expression is essential for cell fate specification in metazoans. Characterizing the transcriptional activity of gene promoters, in time and in space, is therefore a critical step toward understanding complex biological systems. Here we present an in vivo spatiotemporal analysis for approximately 900 predicted C. elegans promoters (approximately 5% of the predicted protein-coding genes), each driving the expression of green fluorescent protein (GFP). Using a flow-cytometer adapted for nematode profiling, we generated 'chronograms', two-dimensional representations of fluorescence intensity along the body axis and throughout development from early larvae to adults. Automated comparison and clustering of the obtained in vivo expression patterns show that genes coexpressed in space and time tend to belong to common functional categories. Moreover, integration of this data set with C. elegans protein-protein interactome data sets enables prediction of anatomical and temporal interaction territories between protein partners. PMID:17486083

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

    PubMed

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

    2015-08-01

    An ideal vascular graft should have the ability to guide the regeneration of neovessels with structure and function similar to those of the native blood vessels. Regeneration of vascular smooth muscle cells (VSMCs) with circumferential orientation within the grafts is crucial for functional vascular reconstruction in vivo. To date, designing and fabricating a vascular graft with well-defined geometric cues to facilitate simultaneously VSMCs infiltration and their circumferential alignment remains a great challenge and scarcely reported in vivo. Thus, we have designed a bi-layered vascular graft, of which the internal layer is composed of circumferentially aligned microfibers prepared by wet-spinning and an external layer composed of random nanofibers prepared by electrospinning. While the internal circumferentially aligned microfibers provide topographic guidance for in vivo regeneration of circumferentially aligned VSMCs, the external random nanofibers can offer enhanced mechanical property and prevent bleeding during and after graft implantation. VSMCs infiltration and alignment within the scaffold was then evaluated in vitro and in vivo. Our results demonstrated that the circumferentially oriented VSMCs and longitudinally aligned ECs were successfully regenerated in vivo after the bi-layered vascular grafts were implanted in rat abdominal aorta. No formation of thrombosis or intimal hyperplasia was observed up to 3 month post implantation. Further, the regenerated neoartery exhibited contraction and relaxation property in response to vasoactive agents. This new strategy may bring cell-free small diameter vascular grafts closer to clinical application. PMID:26001073

  1. 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...

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

    EPA Science Inventory

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

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

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

  3. EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE 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,...

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

    PubMed

    Ruozi, Giulia; Bortolotti, Francesca; Falcione, Antonella; Dal Ferro, Matteo; Ukovich, Laura; Macedo, Antero; Zentilin, Lorena; Filigheddu, Nicoletta; Gortan Cappellari, Gianluca; 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

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

    PubMed Central

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

    2015-01-01

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

  6. Effects of ACL Reconstruction on In-Vivo, Dynamic Knee Function

    PubMed Central

    Tashman, Scott; Araki, Daisuke

    2012-01-01

    Synopsis The purposes of this article are to discuss key factors for assessing joint function, to present some recent findings and to address the future directions for evaluating the function of the ACL-injured/reconstructed knees. Well-designed studies, using state-of-the art tools to assess knee kinematics under in vivo, dynamic, high-loading conditions, are necessary to evaluate the relative performance of different procedures for restoring normal joint motion. PMID:23177461

  7. Comparative lipoplasty analysis of in vivo-treated adipose tissue.

    PubMed

    Rohrich, R J; Morales, D E; Krueger, J E; Ansari, M; Ochoa, O; Robinson, J; Beran, S J

    2000-05-01

    A comparative histologic and chemical analysis was undertaken of adipose tissue treated in vivo with traditional, ultrasound-assisted, and external ultrasound-assisted lipoplasty. A series of six healthy women undergoing elective liposuction according to the superwet technique using a 1:1 infiltration ratio with the estimated quantity of fat to be removed was included in the study. Four separate regions on each patient were treated independently in vivo with traditional liposuction, internal ultrasound-assisted liposuction, or external ultrasound-assisted liposuction for 7 minutes. External massage was used as a control. Four separate specimens of adipose tissue from each patient were assessed for cellular disruption using blinded histologic evaluation. The remainder of tissue was centrifuged to separate the aqueous phase from the cellular components and then spectrophotometrically analyzed for creatinine kinase and glycerol 3-phosphate dehydrogenase activity as markers of cellular disruption. Histologic analysis confirmed 70 to 90 percent cellular disruption with internal ultrasound-assisted liposuction. Suction-assisted and external ultrasound-assisted liposuction showed 5 to 25 percent disruption, whereas massage controls showed only 5 percent. Only internal ultrasound-assisted liposuction showed 5 to 20 percent thermal liquefaction. Absorbance analysis showed creatine kinase activity (sigma units) greatest in ultrasound-exposed tissue. Both external and internal ultrasound-assisted liposuction gave creatine kinase levels 28 to 33 percent greater than suction-assisted liposuction, which varied only 10 percent from controls. Glycerol 3-phosphate dehydrogenase activity was 44 percent greater for internal ultrasound-assisted liposuction than that detected with suction-assisted liposuction. Glycerol 3-phosphate dehydrogenase activity with external ultrasound-assisted liposuction and massage did not vary much from each other, at only 14 percent and 11 percent

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

    PubMed Central

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

    2015-01-01

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

  9. In-vivo visualization and functional characterization of primary somatic neurons

    PubMed Central

    Ma, Chao; Donnelly, David F.; LaMotte, Robert H.

    2010-01-01

    In-vivo electrophysiological recordings from cell bodies of primary sensory neurons are used to determine sensory function but are commonly performed blindly and without access to voltage-(patch-clamp) electrophysiology or optical imaging. We present a procedure to visualize and patch-clamp the neuronal cell body in the dorsal root ganglion, in vivo, manipulate its chemical environment, determine its receptive field properties, and remove it either to obtain subsequent molecular analyses or to gain access to deeper lying cells. This method allows the association of the peripheral transduction capacities of a sensory neuron with the biophysical and chemical characteristics of its cell body. PMID:20558205

  10. Structured Functional Principal Component Analysis

    PubMed Central

    Shou, Haochang; Zipunnikov, Vadim; Crainiceanu, Ciprian M.; Greven, Sonja

    2015-01-01

    Summary Motivated by modern observational studies, we introduce a class of functional models that expand nested and crossed designs. These models account for the natural inheritance of the correlation structures from sampling designs in studies where the fundamental unit is a function or image. Inference is based on functional quadratics and their relationship with the underlying covariance structure of the latent processes. A computationally fast and scalable estimation procedure is developed for high-dimensional data. Methods are used in applications including high-frequency accelerometer data for daily activity, pitch linguistic data for phonetic analysis, and EEG data for studying electrical brain activity during sleep. PMID:25327216

  11. Structured functional principal component analysis.

    PubMed

    Shou, Haochang; Zipunnikov, Vadim; Crainiceanu, Ciprian M; Greven, Sonja

    2015-03-01

    Motivated by modern observational studies, we introduce a class of functional models that expand nested and crossed designs. These models account for the natural inheritance of the correlation structures from sampling designs in studies where the fundamental unit is a function or image. Inference is based on functional quadratics and their relationship with the underlying covariance structure of the latent processes. A computationally fast and scalable estimation procedure is developed for high-dimensional data. Methods are used in applications including high-frequency accelerometer data for daily activity, pitch linguistic data for phonetic analysis, and EEG data for studying electrical brain activity during sleep. PMID:25327216

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

    PubMed

    Prasad, Abhishek; Sanchez, Justin C

    2012-04-01

    Long-term acquisition of high-quality neural recordings is a cornerstone of neuroprosthetic system design. Mitigating the experimental variability of chronically implanted arrays has been a formidable task because the sensor recording sites can be influenced by biotic and abiotic responses. Several studies have implicated changes in electrical interface impedance as a preliminary marker to infer electrode viability. Microelectrode impedance plays an important role in the monitoring of low amplitude and high-resolution extracellular neural signals. In this work, we seek to quantify long-term microelectrode array functionality and derive an impedance-based predictor for electrode functionality that correlates the recording site electrical properties with the functional neuronal recordings in vivo. High temporal resolution metrics of this type would allow one to assess, predict, and improve electrode performance in the future. In a large cohort of animals, we performed daily impedance measurements and neural signal recordings over long periods (up to 21 weeks) of time in rats using tungsten microwire arrays implanted into the somatosensory cortex. This study revealed that there was a time-varying trend in the modulation of impedance that was related to electrode performance. Single units were best detected from electrodes at time points when the electrode entered into the 40-150 KΩ impedance range. This impedance trend was modeled across the full cohort of animals to predict future electrode performance. The model was tested on data from all animals and was able to provide predictions of electrode performance chronically. Insight from this study can be combined with knowledge of electrode materials and histological analysis to provide a more comprehensive predictive model of electrode failure in the future. PMID:22442134

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

    NASA Astrophysics Data System (ADS)

    Prasad, Abhishek; Sanchez, Justin C.

    2012-04-01

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

  14. Adenosine A2A Agonist Improves Lung Function During Ex-vivo Lung Perfusion

    PubMed Central

    Emaminia, Abbas; LaPar, Damien J.; Zhao, Yunge; Steidle, John F.; Harris, David A.; Linden, Joel; Kron, Irving L.; Lau, Christine L.

    2012-01-01

    Background Ex-vivo lung perfusion (EVLP) is a novel technique to assess, and potentially repair marginal lungs that may otherwise be rejected for transplantation. Adenosine has been shown to protect against lung ischemia-reperfusion injury through its A2A receptor. We hypothesized that combining EVLP with adenosine A2A receptor agonist treatment would enhance lung functional quality and increase donor lung usage. Methods Eight bilateral pig lungs were harvested and flushed with cold Perfadex. After 14 hours storage at 4°C, EVLP was performed for 5 hours on two explanted lung groups: 1) Control group lungs (n=4), were perfused with Steen Solution and Dimethyl sulfoxide (DMSO), and 2) treated group lungs (n=4) received 10μM CGS21680, a selective A2A receptor agonist, in a Steen Solution-primed circuit. Lung histology, tissue cytokines, gas analysis and pulmonary function were compared between groups. Results Treated lungs demonstrated significantly less edema as reflected by wet-dry weight ratio (6.6 vs. 5.2, p<0.03) and confirmed by histology. In addition, treated lung demonstrated significantly lower levels of interferon gamma (45.1 vs. 88.5, p<0.05). Other measured tissue cytokines (interleukin (IL) 1 beta, IL-6, and IL-8) were lower in treatment group, but values failed to reach statistical significance. Oxygenation index was improved in the treated group (1.5 vs. 2.3, p<0.01) as well as mean airway pressure (10.3 vs. 13 p<0.009). Conclusions EVLP is a novel and efficient way to assess and optimize lung function and oxygen exchange within donor lungs, and the use of adenosine A2A agonist potentiates its potential. EVLP with the concomitant administration of A2A agonist may enhance donor lung quality and could increase the donor lung pool for transplantation. PMID:22051279

  15. In vivo veritas: using yeast to probe the biological functions of G-quadruplexes

    PubMed Central

    Johnson, Jay E.; Smith, Jasmine S.; Kozak, Marina L.; Johnson, F. Brad

    2008-01-01

    Certain guanine-rich sequences are capable of forming higher order structures known as G-quadruplexes. Moreover, particular genomic regions in a number of highly divergent organisms are enriched for such sequences, raising the possibility that G-quadruplexes form in vivo and affect cellular processes. While G-quadruplexes have been rigorously studied in vitro, whether these structures actually form in vivo and what their roles might be in the context of the cell have remained largely unanswered questions. Recent studies suggest that G-quadruplexes participate in the regulation of such varied processes as telomere maintenance, transcriptional regulation and ribosome biogenesis. Here we review studies aimed at elucidating the in vivo functions of quadruplex structures, with a particular focus on findings in yeast. In addition, we discuss the utility of yeast model systems in the study of the cellular roles of G-quadruplexes. PMID:18331848

  16. A genome scale resource for in vivo tag-based protein function exploration in C. elegans

    PubMed Central

    Sarov, Mihail; Murray, John; Schanze, Kristin; Pozniakovski, Andrei; Niu, Wei; Angermann, Karolin; Hasse, Susanne; Rupprecht, Michaela; Vinis, Elisabeth; Tinney, Matthew; Preston, Elicia; Zinke, Andrea; Enst, Susanne; Teichgraber, Tina; Janette, Judith; Reis, Kadri; Janosch, Stephan; Schloissnig, Siegfried; Ejsmont, Radoslaw K.; Slightam, Cindie; Xu, Xiao; Kim, Stuart K.; Reinke, Valerie; Stewart, A. Francis; Snyder, Michael; Waterston, Robert; Hyman, Anthony A.

    2012-01-01

    Understanding the in vivo dynamics of protein localization and their physical interactions is important for many problems in Biology. To enable systematic protein function interrogation in a multicelluar context, we built a genome-scale transgenic platform for in vivo expression of fluorescent and affinity tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The platform combines computer-assisted transgene design, massively parallel DNA engineering and next generation sequencing to generate a resource of 14637 genomic DNA transgenes, which covers 73% of the proteome. The multipurpose tag used allows any protein of interest to be localized in vivo or affinity purified using standard tag-based assays. We illustrate the utility of the resource by systematic chromatin immunopurification and automated 4D imaging, which produced detailed DNA binding and cell/tissue distribution maps for key transcription factor proteins PMID:22901814

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  18. A protein targeting signal that functions in polarized epithelial cells in vivo.

    PubMed Central

    Ali, S; Hall, J; Hazlewood, G P; Hirst, B H; Gilbert, H J

    1996-01-01

    Eukaryotic membrane-associated polypeptides often contain a glycosylphosphatidylinositol (GPI) anchor that signals the attachment of GPI lipids to these proteins. The GPI anchor can function as a basolateral or apical targeting signal in mammalian cells cultured in vitro, although the function of the GPI anchor in vivo remains to be elucidated. In this study we have evaluated the effect of fusing a GPI anchor sequence to a prokaryotic reporter protein on the cellular location of the polypeptide in polarized epithelial cells of transgenic mice. The bacterial enzyme, when fused to a eukaryotic signal peptide, was secreted through the basolateral membrane of small-intestinal enterocytes; however, when the enzyme was lined to the GPI anchor sequence the polypeptide was redirected to the apical surface of the epithelial cells. These data provide the first direct evidence that the GPI anchor functions as an apical membrane protein sorting signal in polarized epithelial cells in vivo. PMID:8645168

  19. Comparative analysis of SV40 17kT and LT function in vivo demonstrates that LT's C-terminus re-programs hepatic gene expression and is necessary for tumorigenesis in the liver

    PubMed Central

    Comerford, S A; Schultz, N; Hinnant, E A; Klapproth, S; Hammer, R E

    2012-01-01

    Transformation by Simian Virus 40 (SV40) large T antigen (LT) is mediated in large part by its interaction with a variety of cellular proteins at distinct binding domains within LT. While the interaction of LT's N-terminus with the tumor suppressor Rb is absolutely required for LT-dependent transformation, the requirement for the interaction of LT's C-terminus with p53 is less clear and cell- and context-dependent. Here, we report a line of transgenic mice expressing a doxycycline-inducible liver-specific viral transcript that produces abundant 17kT, a naturally occurring SV40 early product that is co-linear with LT for the first 131 amino acids and that binds to Rb, but not p53. Comparative analysis of livers of transgenic mice expressing either 17kT or full length LT demonstrates that 17kT stimulates cell proliferation and induces hepatic hyperplasia but is incapable of inducing hepatic dysplasia or promoting hepatocarcinogenesis. Gene expression profiling demonstrates that 17kT and LT invoke a set of shared molecular signatures consistent with the action of LT's N-terminus on Rb-E2F-mediated control of hepatocyte transcription. However, 17kT also induces a unique set of genes, many of which are known transcriptional targets of p53, while LT actively suppresses them. LT also uniquely deregulates the expression of a subset of genes within the imprinted network and rapidly re-programs hepatocyte gene expression to a more fetal-like state. Finally, we provide evidence that the LT/p53 complex provides a gain-of-function for LT-dependent transformation in the liver, and confirm the absolute requirement for LT's C-terminus for liver tumor development by demonstrating that phosphatase and tensin homolog (PTEN)-deficiency readily cooperates with LT, but not 17kT, for tumorigenesis. These results confirm independent and inter-dependent functions for LT's N- and C-terminus and emphasize differences in the requirements for LT's C-terminus in cell-type dependent

  20. 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

  1. Dissecting the function and assembly of acentriolar microtubule organizing centers in Drosophila cells in vivo.

    PubMed

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

    2015-05-01

    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. 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.

  3. 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.

  4. Development of Chemical Probes for Investigation of Salt-Inducible Kinase Function in Vivo.

    PubMed

    Sundberg, Thomas B; Liang, Yanke; Wu, Huixian; Choi, Hwan Geun; Kim, Nam Doo; Sim, Taebo; Johannessen, Liv; Petrone, Adam; Khor, Bernard; Graham, Daniel B; Latorre, Isabel J; Phillips, Andrew J; Schreiber, Stuart L; Perez, Jose; Shamji, Alykhan F; Gray, Nathanael S; Xavier, Ramnik J

    2016-08-19

    Salt-inducible kinases (SIKs) are promising therapeutic targets for modulating cytokine responses during innate immune activation. The study of SIK inhibition in animal models of disease has been limited by the lack of selective small-molecule probes suitable for modulating SIK function in vivo. We used the pan-SIK inhibitor HG-9-91-01 as a starting point to develop improved analogs, yielding a novel probe 5 (YKL-05-099) that displays increased selectivity for SIKs versus other kinases and enhanced pharmacokinetic properties. Well-tolerated doses of YKL-05-099 achieve free serum concentrations above its IC50 for SIK2 inhibition for >16 h and reduce phosphorylation of a known SIK substrate in vivo. While in vivo active doses of YKL-05-099 recapitulate the effects of SIK inhibition on inflammatory cytokine responses, they did not induce metabolic abnormalities observed in Sik2 knockout mice. These results identify YKL-05-099 as a useful probe to investigate SIK function in vivo and further support the development of SIK inhibitors for treatment of inflammatory disorders. PMID:27224444

  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. Uptake of postprandial lipoproteins into bone in vivo: impact on osteoblast function.

    PubMed

    Niemeier, Andreas; Niedzielska, Dagmara; Secer, Rukiye; Schilling, Arndt; Merkel, Martin; Enrich, Carlos; Rensen, Patrick C N; Heeren, Joerg

    2008-08-01

    Dietary lipids and lipophilic vitamins are transported by postprandial lipoproteins and are required for bone metabolism. Despite that, it remains unknown whether bone cells are involved in the uptake of circulating postprandial lipoproteins in vivo. The current study was performed to investigate a putative participation of bone in the systemic postprandial lipoprotein metabolism in mice, to identify potentially involved cell type populations and to analyze whether lipoprotein uptake affects bone function in vivo. As a model for the postprandial state, chylomicron remnants (CR) were injected intravenously into mice. Next to the liver and compared to other organs, bone appeared to be the second most important organ for the clearance of radiolabeled CR particles from the circulation in vivo. In addition, uptake of radiolabeled CR by primary murine osteoblasts and hepatocytes was quantified to be in a similar range in vitro. A complementary approach with fluorescently labeled CR and immunohistochemical staining for apoE proved that intact CR particles were taken up into bone and liver. Electron microscopy localization studies of bone sections revealed CR uptake into sinusoidal endothelial cells, macrophages and osteoblasts. The relative amount of radiolabeled CR uptake into femoral cortical bone, representing predominantly osteoblasts, and bone marrow, representing predominantly non-osteoblast cells, was within the same range. Most importantly, the injection of vitamin K1-enriched CR resulted in an increase of the degree of osteocalcin carboxylation in vivo while total osteocalcin concentrations remained unaffected, giving functional proof that osteoblasts process CR in vivo. In conclusion, here we demonstrate that bone is involved in the postprandial lipoprotein metabolism in mice. Osteoblasts participate in CR clearance from the circulation, which has a direct impact on the secretory function of osteoblasts. PMID:18538644

  7. 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.

  8. In vivo analysis of Caenorhabditis elegans noncoding RNA promoter motifs

    PubMed Central

    Li, Tiantian; He, Housheng; Wang, Yunfei; Zheng, Haixia; Skogerbø, Geir; Chen, Runsheng

    2008-01-01

    Background Noncoding RNAs (ncRNAs) play important roles in a variety of cellular processes. Characterizing the transcriptional activity of ncRNA promoters is therefore a critical step toward understanding the complex cellular roles of ncRNAs. Results Here we present an in vivo transcriptional analysis of three C. elegans ncRNA upstream motifs (UM1-3). Transcriptional activity of all three motifs has been demonstrated, and mutational analysis revealed differential contributions of different parts of each motif. We showed that upstream motif 1 (UM1) can drive the expression of green fluorescent protein (GFP), and utilized this for detailed analysis of temporal and spatial expression patterns of 5 SL2 RNAs. Upstream motifs 2 and 3 do not drive GFP expression, and termination at consecutive T runs suggests transcription by RNA polymerase III. The UM2 sequence resembles the tRNA promoter, and is actually embedded within its own short-lived, primary transcript. This is a structure which is also found at a few plant and yeast loci, and may indicate an evolutionarily very old dicistronic transcription pattern in which a tRNA serves as a promoter for an adjacent snoRNA. Conclusion The study has demonstrated that the three upstream motifs UM1-3 have promoter activity. The UM1 sequence can drive expression of GFP, which allows for the use of UM1::GFP fusion constructs to study temporal-spatial expression patterns of UM1 ncRNA loci. The UM1 loci appear to act in concert with other upstream sequences, whereas the transcriptional activities of the UM2 and UM3 are confined to the motifs themselves. PMID:18680611

  9. In Vivo Function of Tryptophans in the Arabidopsis UV-B Photoreceptor UVR8[W

    PubMed Central

    O’Hara, Andrew; Jenkins, Gareth I.

    2012-01-01

    Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor specifically for UV-B light that initiates photomorphogenic responses in plants. UV-B exposure causes rapid conversion of UVR8 from dimer to monomer, accumulation in the nucleus, and interaction with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), which functions with UVR8 in UV-B responses. Studies in yeast and with purified UVR8 implicate several tryptophan amino acids in UV-B photoreception. However, their roles in UV-B responses in plants, and the functional significance of all 14 UVR8 tryptophans, are not known. Here we report the functions of the UVR8 tryptophans in vivo. Three tryptophans in the β-propeller core are important in maintaining structural stability and function of UVR8. However, mutation of three other core tryptophans and four at the dimeric interface has no apparent effect on function in vivo. Mutation of three tryptophans implicated in UV-B photoreception, W233, W285, and W337, impairs photomorphogenic responses to different extents. W285 is essential for UVR8 function in plants, whereas W233 is important but not essential for function, and W337 has a lesser role. Ala mutants of these tryptophans appear monomeric and constitutively bind COP1 in plants, but their responses indicate that monomer formation and COP1 binding are not sufficient for UVR8 function. PMID:23012433

  10. 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

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

    DOE PAGESBeta

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

    2015-03-11

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

  12. 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.

  13. 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

  14. Transcriptional Regulation of Rod Photoreceptor Homeostasis Revealed by In Vivo NRL Targetome Analysis

    PubMed Central

    Hao, Hong; Kim, Douglas S.; Klocke, Bernward; Johnson, Kory R.; Cui, Kairong; Gotoh, Norimoto; Zang, Chongzhi; Gregorski, Janina; Gieser, Linn; Peng, Weiqun; Fann, Yang; Seifert, Martin; Zhao, Keji; Swaroop, Anand

    2012-01-01

    A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP–Seq) data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP–Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP–Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis. PMID:22511886

  15. Generalized functional extended redundancy analysis.

    PubMed

    Hwang, Heungsun; Suk, Hye Won; Takane, Yoshio; Lee, Jang-Han; Lim, Jooseop

    2015-03-01

    Functional extended redundancy analysis (FERA) was recently developed to integrate data reduction into functional linear models. This technique extracts a component from each of multiple sets of predictor data in such a way that the component accounts for the maximum variance of response data. Moreover, it permits predictor and/or response data to be functional. FERA can be of use in describing overall characteristics of each set of predictor data and in summarizing the relationships between predictor and response data. In this paper, we extend FERA into the framework of generalized linear models (GLM), so that it can deal with response data generated from a variety of distributions. Specifically, the proposed method reduces each set of predictor functions to a component and uses the component for explaining exponential-family responses. As in GLM, we specify the random, systematic, and link function parts of the proposed method. We develop an iterative algorithm to maximize a penalized log-likelihood criterion that is derived in combination with a basis function expansion approach. We conduct two simulation studies to investigate the performance of the proposed method based on synthetic data. In addition, we apply the proposed method to two examples to demonstrate its empirical usefulness. PMID:24271507

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

    NASA Astrophysics Data System (ADS)

    Shor, Erez; Shoham, Shy; Levenberg, Shulamit

    2016-03-01

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

  17. Functional Studies of the Carboxy-Terminal Repeat Domain of Drosophila RNA Polymerase II in Vivo

    PubMed Central

    Brickey, W. J.; Greenleaf, A. L.

    1995-01-01

    To understand the in vivo function of the unique and conserved carboxy-terminal repeat domain (CTD) of RNA polymerase II largest subunit (RpII215), we have studied RNA polymerase II biosynthesis, activity and genetic function in Drosophila RpII215 mutants that possessed all (C4), half (W81) or none (IIt) of the CTD repeats. We have discovered that steady-state mRNA levels from transgenes encoding a fully truncated, CTD-less subunit (IIt) are essentially equal to wild-type levels, whereas the levels of the CTD-less subunit itself and the amount of polymerase harboring it (Pol IIT) are significantly lower than wild type. In contrast, for the half-CTD mutant (W81), steady-state mRNA levels are somewhat lower than for wild type or IIt, while W81 subunit and polymerase amounts are much less than wild type. Finally, we have tested genetically the ability of CTD mutants to complement (rescue) partially functional RpII215 alleles and have found that IIt fails to complement whereas W81 complements partially to completely. These results suggest that removal of the entire CTD renders polymerase completely defective in vivo, whereas eliminating half of the CTD results in a polymerase with significant in vivo activity. PMID:7498740

  18. 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.

  19. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2010-04-01

    In this paper, we report the use of near-infrared (NIR)-emitting alloyed quantum dots (QDs) as efficient optical probes for high contrast in vivo imaging of tumors. Alloyed CdTe1 - xSex/CdS QDs were prepared in the non-aqueous phase using the hot colloidal synthesis approach. Water dispersion of the QDs were accomplished by their encapsulation within polyethyleneglycol (PEG)-grafted phospholipid micelles. For tumor-specific delivery in vivo, the micelle-encapsulated QDs were conjugated with the cyclic arginine-glycine-aspartic acid (cRGD) peptide, which targets the αvβ3 integrins overexpressed in the angiogenic tumor vasculatures. Using in vivo NIR optical imaging of mice bearing pancreatic cancer xenografts, implanted both subcutaneously and orthotopically, we have demonstrated that systemically delivered cRGD-conjugated QDs, but not the unconjugated ones, can efficiently target and label the tumors with high signal-to-noise ratio. Histopathological analysis of major organs of the treated mice showed no evidence of systemic toxicity associated with these QDs. These experiments suggest that cRGD-conjugated NIR QDs can serve as safe and efficient probes for optical bioimaging of tumors in vivo. Furthermore, by co-encapsulating these QDs and anticancer drugs within these micelles, we have demonstrated a promising theranostic, nanosized platform for both cancer imaging and therapy.

  2. 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

  3. Alteration of canine left ventricular diastolic function by intravenous anesthetics in vivo. Ketamine and propofol.

    PubMed

    Pagel, P S; Schmeling, W T; Kampine, J P; Warltier, D C

    1992-03-01

    Diastolic function has been shown to influence overall cardiac performance significantly, but the effect of intravenous anesthetics on diastolic function has not been previously characterized in vivo. The effects of ketamine and propofol on two indices of left ventricular diastolic function were examined in chronically instrumented dogs. Because autonomic nervous system function may significantly influence the systemic hemodynamic actions produced by intravenous anesthetics in vivo, experiments were performed in the presence of pharmacologic blockade of the autonomic nervous system. Two groups comprising a total of 14 experiments were performed using 7 dogs instrumented for measurement of aortic and left ventricular pressure, the maximum rate of increase of left ventricular pressure (dP/dt), subendocardial segment length, and cardiac output. Systemic hemodynamics and diastolic function were recorded and evaluated in the conscious state and after a 20-min equilibration at 25-, 50-, and 100-mg.kg-1.h-1 infusion doses of ketamine or propofol. Ventricular relaxation was described using the time constant of isovolumetric relaxation (tau) assuming a nonzero asymptote of ventricular pressure decay. Regional chamber stiffness, an index of passive ventricular filling, was described using an exponential equation relating segment length to ventricular pressure between minimum ventricular pressure and the onset of atrial systole.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1539854

  4. Transcription factor YY1 functions as a PcG protein in vivo.

    PubMed

    Atchison, Lakshmi; Ghias, Ayesha; Wilkinson, Frank; Bonini, Nancy; Atchison, Michael L

    2003-03-17

    Polycomb group (PcG) proteins function as high molecular weight complexes that maintain transcriptional repression patterns during embryogenesis. The vertebrate DNA binding protein and transcriptional repressor, YY1, shows sequence homology with the Drosophila PcG protein, pleiohomeotic (PHO). YY1 might therefore be a vertebrate PcG protein. We used Drosophila embryo and larval/imaginal disc transcriptional repression systems to determine whether YY1 repressed transcription in a manner consistent with PcG function in vivo. YY1 repressed transcription in Drosophila, and this repression was stable on a PcG-responsive promoter, but not on a PcG-non-responsive promoter. PcG mutants ablated YY1 repression, and YY1 could substitute for PHO in repressing transcription in wing imaginal discs. YY1 functionally compensated for loss of PHO in pho mutant flies and partially corrected mutant phenotypes. Taken together, these results indicate that YY1 functions as a PcG protein. Finally, we found that YY1, as well as Polycomb, required the co-repressor protein CtBP for repression in vivo. These results provide a mechanism for recruitment of vertebrate PcG complexes to DNA and demonstrate new functions for YY1. PMID:12628927

  5. In vivo functional microangiography by visible-light optical coherence tomography

    PubMed Central

    Yi, Ji; Chen, Siyu; Backman, Vadim; Zhang, Hao F.

    2014-01-01

    Although hemoglobin oxygen saturation (sO2) in the microvasculature is an essential physiological parameter of local tissue functions, non-invasive measurement of microvascular sO2 is still challenging. Here, we demonstrated that visible-light optical coherence tomography (vis-OCT) can simultaneously provide three-dimensional anatomical tissue morphology, visualize microvasculature at the capillary level, and measure sO2 from the microvasculature in vivo. We utilized speckle contrast caused by the moving blood cells to enhance microvascular imaging. We applied a series of short-time inverse Fourier transforms to obtain the spectroscopic profile of blood optical attenuation, from which we quantified sO2. We validated the sO2 measurement in mouse ears in vivo through hypoxia and hyperoxia challenges. We further demonstrated that vis-OCT can continuously monitor dynamic changes of microvascular sO2. PMID:25360376

  6. Dual-selection for evolution of in vivo functional aptazymes as riboswitch parts.

    PubMed

    Goler, Jonathan A; Carothers, James M; Keasling, Jay D

    2014-01-01

    Both synthetic biology and metabolic engineering are aided by the development of genetic control parts. One class of riboswitch parts that has great potential for sensing and regulation of protein levels is aptamer-coupled ribozymes (aptazymes). These devices are comprised of an aptamer domain selected to bind a particular ligand, a ribozyme domain, and a communication module that regulates the ribozyme activity based on the state of the aptamer. We describe a broadly applicable method for coupling a novel, newly selected aptamer to a ribozyme to generate functional aptazymes via in vitro and in vivo selection. To illustrate this approach, we describe experimental procedures for selecting aptazymes assembled from aptamers that bind p-amino-phenylalanine and a hammerhead ribozyme. Because this method uses selection, it does not rely on sequence-specific design and thus should be generalizable for the generation of in vivo operational aptazymes that respond to any targeted molecules. PMID:24549623

  7. B Plant function analysis report

    SciTech Connect

    Lund, D.P.; B Plant Working Group

    1995-09-01

    The document contains the functions, function definitions, function interfaces, function interface definitions, Input Computer Automated Manufacturing Definition (IDEFO) diagrams, and a function hierarchy chart that describe what needs to be performed to deactivate B Plant.

  8. In vivo Function and Membrane Binding Properties are Correlated for Escherichia coli LamB Signal Peptides

    NASA Astrophysics Data System (ADS)

    Briggs, Martha S.; Gierasch, Lila M.; Zlotnick, Adam; Lear, James D.; Degrado, William F.

    1985-05-01

    Wild-type and pseudorevertant signal peptides of the lamB gene product of Escherichia coli interact with lipid systems whereas a nonfunctional deletion mutant signal peptide does not. This conclusion is based on (i) interaction of synthetic signal peptides with a lipid monolayer-water surface, (ii) conformational changes induced by presence of lipid vesicles in an aqueous solution of signal peptide, and (iii) capacities of the peptides to promote vesicle aggregation. Analysis of the signal sequences and previous conformational studies suggest that these lipid interaction properties may be attributable to the tendency of the functional signal peptides to adopt α -helical conformations. Although the possibility of direct interaction between the signal peptide and membrane lipids during protein secretion is controversial, the results suggest that conformationally related amphiphilicity and consequent membrane affinity of signal sequences are important for function in vivo.

  9. Critical role of tissue mast cells in controlling long-term glucose sensor function in vivo.

    PubMed

    Klueh, Ulrike; Kaur, Manjot; Qiao, Yi; Kreutzer, Donald L

    2010-06-01

    Little is known about the specific cells, mediators and mechanisms involved in the loss of glucose sensor function (GSF) in vivo. Since mast cells (MC) are known to be key effector cells in inflammation and wound healing, we hypothesized that MC and their products are major contributors to the skin inflammation and wound healing that controls GSF at sites of sensor implantation. To test this hypothesis we utilized a murine model of continuous glucose monitoring (CGM) in vivo in both normal C57BL/6 mice (mast cell sufficient), as well as mast cell deficient B6.Cg-Kit(W-sh)/HNihrJaeBsmJ (Sash) mice over a 28 day CGM period. As expected, both strains of mice displayed excellent CGM for the first 7 days post sensor implantation (PSI). CGM in the mast cell sufficient C57BL/6 mice was erratic over the remaining 21 days PSI. CGM in the mast cell deficient Sash mice displayed excellent sensor function for the entire 28 day of CGM. Histopathologic evaluation of implantation sites demonstrated that tissue reactions in Sash mice were dramatically less compared to the reactions in normal C57BL/6 mice. Additionally, mast cells were also seen to be consistently associated with the margins of sensor tissue reactions in normal C57BL/6 mice. Finally, direct injection of bone marrow derived mast cells at sites of sensor implantation induced an acute and dramatic loss of sensor function in both C57BL/6 and Sash mice. These results demonstrate the key role of mast cells in controlling glucose sensor function in vivo. PMID:20226521

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

    PubMed Central

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

    2008-01-01

    Purpose To relate in vivo microscopic retinal changes to visual function in patients who have various forms of retinal dystrophy. Methods The UC Davis Adaptive Optics (AO) fundus camera was used to acquire in vivo retinal images at the cellular level. Visual function tests consisting of visual fields, multifocal electroretinography (mfERG), and contrast sensitivity were measured in all subjects by using stimuli that were coincident with areas imaged. Five patients with different forms of retinal dystrophy and three control subjects were recruited. Cone densities were quantified for all retinal images. Results In all images of diseased retinas, there were extensive areas of dark space between groups of photoreceptors, where no cone photoreceptors were evident. These irregular features were not seen in healthy retinas, but were apparent in patients with retinal dystrophy. There were significant correlations between functional vision losses and the extent to which these irregularities, quantified by cone density, occurred in retinal images. Conclusions AO fundus imaging is a reliable technique for assessing and quantifying the changes in the photoreceptor layer as disease progresses. Furthermore, this technique can be useful in cases where visual function tests provide borderline or ambiguous results, as it allows visualization of individual photoreceptors. PMID:16639019

  11. Functional Multiple-Set Canonical Correlation Analysis

    ERIC Educational Resources Information Center

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

    2012-01-01

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

  12. 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. Occupational Functionality: A Concept Analysis.

    PubMed

    Combs, Bryan; Heaton, Karen

    2016-08-01

    Occupational health nursing has evolved since the late 19th century and, with the inclusion of advanced practice nursing, has become essential to the health and safety of workers. A key component of the knowledge required of advanced practice occupational health nurses is an understanding of what it means for workers to be fit for duty The definition or concept of being fit for duty varies depending on the point-of-view of the health care provider. Health care providers across all professions must have a consistent understanding of what it means to be fit for duty Literature shows that professions and specialties that often collaborate have varying ideas about what it means to be fit for duty These differences highlight the need for a consistent concept that can be used across professions, is holistic, and incorporates other concepts critical to all points of view. To better understand fit for duty, a concept analysis, using the Walker and Avant framework, focused on the concept of occupational functionality (OF). Occupational functionality is best defined as the qualities of being suited to serve an occupational purpose efficiently and effectively within the physical, occupational, environmental, and psychological demands of a unique work setting. This concept analysis offers an initial step in understanding fit for duty and gives health care providers a concept that can be used across disciplines. PMID:27462030

  14. Dopamine impairs functional integrity of rat hepatocytes through nuclear factor kappa B activity modulation: An in vivo, ex vivo, and in vitro study.

    PubMed

    Sun, Cheuk-Kwan; Kao, Ying-Hsien; Lee, Po-Huang; Wu, Ming-Chang; Chen, Kun-Cho; Lin, Yu-Chun; Tsai, Ming-Shian; Chen, Po-Han

    2015-12-01

    Dopamine (DA) is commonly used to maintain the hemodynamic stability of brain-dead donors despite its controversial effects on organ functions. This study aimed at examining the hemodynamic effect of DA in a rat brain-dead model in vivo, alteration of hepatocyte integrity in liver grafts after ex vivo preservation, and changes in cultured clone-9 hepatocytes including cellular viability, cell cycle, apoptotic regulators, and lipopolysaccharide (LPS)-stimulated nuclear factor kappa B (NF-κB) signaling machinery. Although in vivo findings demonstrated enhanced portal venous blood flow and hepatic microcirculatory perfusion after DA infusion, no apparent advantage was noted in preserving hepatocyte integrity ex vivo. In vitro, prolonged exposure to high-dose DA reduced proliferation and induced G1 growth arrest of clone-9 hepatocytes with concomitant decreases in B cell lymphoma 2 (BCL2)/B cell lymphoma 2-associated X protein (BAX) and heat shock protein 70/BAX protein ratios and intracellular NF-κB p65. Moreover, DA pretreatment suppressed LPS-elicited inhibitor of κBα phosphorylation and subsequent NF-κB nuclear translocation, suggesting that DA may down-regulate NF-κB signaling, thereby reducing expression of antiapoptotic regulators, such as BCL2. In conclusion, despite augmentation of hepatic perfusion, DA infusion failed to preserve hepatocyte integrity both in vivo and ex vivo. In vitro findings demonstrated that high-dose DA may hamper the function of NF-κB signaling machinery and eventually undermine functional integrity of hepatocytes in liver grafts. PMID:26421799

  15. 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 ...

  16. 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

  17. PEG-mediated synthesis of highly dispersive multifunctional superparamagnetic nanoparticles: their physicochemical properties and function in vivo.

    PubMed

    Sun, Conroy; Du, Kim; Fang, Chen; Bhattarai, Narayan; Veiseh, Omid; Kievit, Forrest; Stephen, Zachary; Lee, Donghoon; Ellenbogen, Richard G; Ratner, Buddy; Zhang, Miqin

    2010-04-27

    Multifunctional superparamagnetic nanoparticles have been developed for a wide range of applications in nanomedicine, such as serving as tumor-targeted drug carriers and molecular imaging agents. To function in vivo, the development of these novel materials must overcome several challenging requirements including biocompatibility, stability in physiological solutions, nontoxicity, and the ability to traverse biological barriers. Here we report a PEG-mediated synthesis process to produce well-dispersed, ultrafine, and highly stable iron oxide nanoparticles for in vivo applications. Utilizing a biocompatible PEG coating bearing amine functional groups, the produced nanoparticles serve as an effective platform with the ability to incorporate a variety of targeting, therapeutic, or imaging ligands. In this study, we demonstrated tumor-specific accumulation of these nanoparticles through both magnetic resonance and optical imaging after conjugation with chlorotoxin, a peptide with high affinity toward tumors of the neuroectodermal origin, and Cy5.5, a near-infrared fluorescent dye. Furthermore, we performed preliminary biodistribution and toxicity assessments of these nanoparticles in wild-type mice through histological analysis of clearance organs and hematology assay, and the results demonstrated the relative biocompatibility of these nanoparticles. PMID:20232826

  18. 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

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

    PubMed

    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

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

    PubMed

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

    2016-01-01

    Treatment for heart disease, the leading cause of death in the world, has progressed little for several decades. Here we develop a protein engineering approach to directly tune in vivo cardiac contractility by tailoring the ability of the heart to respond to the Ca(2+) signal. Promisingly, our smartly formulated Ca(2+)-sensitizing TnC (L48Q) enhances heart function without any adverse effects that are commonly observed with positive inotropes. In a myocardial infarction (MI) model of heart failure, expression of TnC L48Q before the MI preserves cardiac function and performance. Moreover, expression of TnC L48Q after the MI therapeutically enhances cardiac function and performance, without compromising survival. We demonstrate engineering TnC can specifically and precisely modulate cardiac contractility that when combined with gene therapy can be employed as a therapeutic strategy for heart disease. PMID:26908229

  1. Biomechanical regulation of vascular smooth muscle cell functions: from in vitro to in vivo understanding

    PubMed Central

    Qiu, Juhui; Zheng, Yiming; Hu, Jianjun; Liao, Donghua; Gregersen, Hans; Deng, Xiaoyan; Fan, Yubo; Wang, Guixue

    2014-01-01

    Vascular smooth muscle cells (VSMCs) have critical functions in vascular diseases. Haemodynamic factors are important regulators of VSMC functions in vascular pathophysiology. VSMCs are physiologically active in the three-dimensional matrix and interact with the shear stress sensor of endothelial cells (ECs). The purpose of this review is to illustrate how haemodynamic factors regulate VSMC functions under two-dimensional conditions in vitro or three-dimensional co-culture conditions in vivo. Recent advances show that high shear stress induces VSMC apoptosis through endothelial-released nitric oxide and low shear stress upregulates VSMC proliferation and migration through platelet-derived growth factor released by ECs. This differential regulation emphasizes the need to construct more actual environments for future research on vascular diseases (such as atherosclerosis and hypertension) and cardiovascular tissue engineering. PMID:24152813

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

    PubMed Central

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

    2016-01-01

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

  3. TYK2 Kinase Activity Is Required for Functional Type I Interferon Responses In Vivo

    PubMed Central

    Prchal-Murphy, Michaela; Semper, Christian; Lassnig, Caroline; Wallner, Barbara; Gausterer, Christian; Teppner-Klymiuk, Ingeborg; Kobolak, Julianna; Müller, Simone; Kolbe, Thomas; Karaghiosoff, Marina; Dinnyés, Andras; Rülicke, Thomas; Leitner, Nicole R.; Strobl, Birgit; Müller, Mathias

    2012-01-01

    Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2K923E) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein’s stability. An inhibitory function was only observed upon over-expression of TYK2K923E in vitro. Tyk2K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors. PMID:22723949

  4. Fatigue alters in vivo function within and between limb muscles during locomotion

    PubMed Central

    Higham, Timothy E.; Biewener, Andrew A.

    2008-01-01

    Muscle fatigue, a reduction in force as a consequence of exercise, is an important factor for any animal that moves, and can result from both peripheral and/or central mechanisms. Although much is known about whole-limb force generation and activation patterns in fatigued muscles under sustained isometric contractions, little is known about the in vivo dynamics of limb muscle function in relation to whole-body fatigue. Here we show that limb kinematics and contractile function in the lateral (LG) and medial (MG) gastrocnemius of helmeted guineafowl (Numida meleagris) are significantly altered following fatiguing exercise at 2 m s−1 on an inclined treadmill. The two most significant findings were that the variation in muscle force generation, measured directly from the muscles' tendons, increased significantly with fatigue, and fascicle shortening in the proximal MG, but not the distal MG, decreased significantly with fatigue. We suggest that the former is a potential mechanism for decreased stability associated with fatigue. The region-specific alteration of fascicle behaviour within the MG as a result of fatigue suggests a complex response to fatigue that probably depends on muscle–aponeurosis and tendon architecture not previously explored. These findings highlight the importance of studying the integrative in vivo dynamics of muscle function in response to fatigue. PMID:19129096

  5. Fatigue alters in vivo function within and between limb muscles during locomotion.

    PubMed

    Higham, Timothy E; Biewener, Andrew A

    2009-03-22

    Muscle fatigue, a reduction in force as a consequence of exercise, is an important factor for any animal that moves, and can result from both peripheral and/or central mechanisms. Although much is known about whole-limb force generation and activation patterns in fatigued muscles under sustained isometric contractions, little is known about the in vivo dynamics of limb muscle function in relation to whole-body fatigue. Here we show that limb kinematics and contractile function in the lateral (LG) and medial (MG) gastrocnemius of helmeted guineafowl (Numida meleagris) are significantly altered following fatiguing exercise at 2ms-1 on an inclined treadmill. The two most significant findings were that the variation in muscle force generation, measured directly from the muscles' tendons, increased significantly with fatigue, and fascicle shortening in the proximal MG, but not the distal MG, decreased significantly with fatigue. We suggest that the former is a potential mechanism for decreased stability associated with fatigue. The region-specific alteration of fascicle behaviour within the MG as a result of fatigue suggests a complex response to fatigue that probably depends on muscle-aponeurosis and tendon architecture not previously explored. These findings highlight the importance of studying the integrative in vivo dynamics of muscle function in response to fatigue. PMID:19129096

  6. In vivo left ventricular function and collagen expression in aldosterone/salt-induced hypertension.

    PubMed

    Ramirez-Gil, J F; Delcayre, C; Robert, V; Wassef, M; Trouve, P; Mougenot, N; Charlemagne, D; Lechat, P

    1998-12-01

    Cardiac fibrosis is linked to aldosterone-induced hypertension, but the effects on in vivo left ventricular (LV) function are not established. We studied the relations between in vivo LV function and aldosterone/salt cardiac fibrosis. Adult guinea pigs (GPs) were treated for 3 months with an aldosterone infusion and high-salt diet. This treatment induced arterial hypertension (+35%) and moderate LV hypertrophy (LVH; +60%) without right ventricular (RV) hypertrophy. Echo-Doppler LV assessment demonstrated unaltered cardiac output, stroke volume, or LV relaxation. Type I collagen messenger RNA (mRNA) was significantly increased in both ventricles (LV, +48%; RV, +77%) and accompanied by a significant increase in total collagen deposition (LV, from 0.52% in controls to 4.4% in treated GPs; RV, from 0.82 to 5.5% in treated GPs). Plasma norepinephrine levels increased 2.6-fold (p < 0.01) and correlated with the increase in collagen deposition in both ventricles. Collagen content was not correlated with hypertension or LVH. We conclude that aldosterone administration induces cardiac collagen accumulation and a sympathetic stimulation, which might preserve systolic and diastolic function. PMID:9869498

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

    PubMed

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

    2016-03-14

    The goal of this study is to summarize the current knowledge on the effects of one of the essential metals, copper (Cu) on the reproductive system. The development of past four decades addressing effects of Cu on reproductive organs is reviewed. The most relevant data obtained from in vivo and in vitro experiments performed on humans and other mammals, including effects of copper nanoparticles (CuNPs) on the reproductive functions are presented. Short term Cu administration has been found to exert deleterious effect on intracellular organelles of rat ovarian cells in vivo. In vitro administration in porcine ovarian granulosa cells releases insulin-like growth factor (IGF-I), steroid hormone progesterone (P(4)), and induces expression of peptides related to proliferation and apoptosis. Adverse effect of Cu on male reproductive functions has been indicated by the decrease in spermatozoa parameters such as concentration, viability and motility. Copper nanoparticles are capable of generating oxidative stress in vitro thereby leading to reproductive toxicity. Toxic effect of CuNPs has been evident more in male mice than in females. Even though further investigations are necessary to arrive at a definitive conclusion, Cu notably influences the reproductive functions by interfering with both male and female reproductive systems and also hampers embryo development in dose-dependent manner. PMID:26596322

  8. 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

  9. 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

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

    PubMed

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

    2016-09-01

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

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

    PubMed

    Schlinger, Henry D; Normand, Matthew P

    2013-01-01

    In this essay, we note that although Iwata, Dorsey, Slifer, Bauman, and Richman (1982) established the standard framework for conducting functional analyses of problem behavior, the term functional analysis was probably first used in behavior analysis by B. F. Skinner in 1948. We also remind readers that a functional analysis is really an experimental analysis, words that were contained in the title of Skinner's first book, The Behavior of Organisms: An Experimental Analysis (1938). We further describe how Skinner initially applied the concept of functional analysis to an understanding of verbal behavior, and we suggest that the same tactic be applied to the verbal behavior of behavior analysts, in the present case, to the term functional analysis. PMID:24114100

  12. Isolation of Cardiomyocytes and Cardiofibroblasts for Ex Vivo Analysis.

    PubMed

    Mbogo, George Williams; Nedeva, Christina; Puthalakath, Hamsa

    2016-01-01

    Heart failure (HF) is a common clinical endpoint to several underlying causes including aging, hypertension, stress, and cardiomyopathy. It is characterized by a significant decline in the cardiac output. Cardiomyocytes are terminally differentiated cells and therefore, apoptotic death due to beta adrenergic (β-AR) signaling contributes to high attrition rate of these cells. Past treatments of HF offer some survival benefit to patients (e.g., the beta blockers), but at the expense of blocking the compensatory beta-adrenergic signaling in surviving cells. One prerequisite for developing new therapeutics is to be able to grow cardiomyocytes ex vivo, and test their apoptotic response to drugs. Here we describe methods for isolation and culturing of neonatal and adult calcium tolerant cardiomyocytes. Similarly, cardiofibroblasts can also be isolated using the same protocol and subsequently, immortalized with SV40 T-Antigen for ex vivo studies. PMID:27108436

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

    SciTech Connect

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

    1982-01-01

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

  14. 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

  15. 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.

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

    PubMed Central

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

    2008-01-01

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

  17. 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 α-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

  18. Translation initiation factors are not required for Dicistroviridae IRES function in vivo

    PubMed Central

    Deniz, Nilsa; Lenarcic, Erik M.; Landry, Dori M.; Thompson, Sunnie R.

    2009-01-01

    The cricket paralysis virus (CrPV) intergenic region (IGR) internal ribosome entry site (IRES) uses an unusual mechanism of initiating translation, whereby the IRES occupies the P-site of the ribosome and the initiating tRNA enters the A-site. In vitro experiments have demonstrated that the CrPV IGR IRES is able to bind purified ribosomes and form 80S complexes capable of synthesizing small peptides in the absence of any translation initiation factors. These results suggest that initiation by this IRES is factor-independent. To determine whether the IGR IRES functions in the absence of initiation factors in vivo, we assayed IGR IRES activity in various yeast strains harboring mutations in canonical translation initiation factors. We used a dicistronic reporter assay in yeast to determine whether the CrPV IGR IRES is able to promote translation sufficient to support growth in the presence of various deletions or mutations in translation initiation factors. Using this assay, we have previously shown that the CrPV IGR IRES functions efficiently in yeast when ternary complexes (eIF2•GTP•initiator tRNAmet) are reduced. Here, we demonstrate that the CrPV IGR IRES activity does not require the eukaryotic initiation factors eIF4G1 or eIF5B, and it is enhanced when eIF2B, the eIF3b subunit of eIF3, or eIF4E are impaired. Taken together, these data support a model in which the CrPV IGR IRES is capable of initiating protein synthesis in the absence of any initiation factors in vivo, and suggests that the CrPV IGR IRES initiates translation by directly recruiting the ribosomal subunits in vivo. PMID:19299549

  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 Analysis and Treatment of Nail Biting

    ERIC Educational Resources Information Center

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

    2008-01-01

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

  1. Small Molecule Agonists of Cell Adhesion Molecule L1 Mimic L1 Functions In Vivo.

    PubMed

    Kataria, Hardeep; Lutz, David; Chaudhary, Harshita; Schachner, Melitta; Loers, Gabriele

    2016-09-01

    Lack of permissive mechanisms and abundance of inhibitory molecules in the lesioned central nervous system of adult mammals contribute to the failure of functional recovery after injury, leading to severe disabilities in motor functions and pain. Peripheral nerve injury impairs motor, sensory, and autonomic functions, particularly in cases where nerve gaps are large and chronic nerve injury ensues. Previous studies have indicated that the neural cell adhesion molecule L1 constitutes a viable target to promote regeneration after acute injury. We screened libraries of known drugs for small molecule agonists of L1 and evaluated the effect of hit compounds in cell-based assays in vitro and in mice after femoral nerve and spinal cord injuries in vivo. We identified eight small molecule L1 agonists and showed in cell-based assays that they stimulate neuronal survival, neuronal migration, and neurite outgrowth and enhance Schwann cell proliferation and migration and myelination of neurons in an L1-dependent manner. In a femoral nerve injury mouse model, enhanced functional regeneration and remyelination after application of the L1 agonists were observed. In a spinal cord injury mouse model, L1 agonists improved recovery of motor functions, being paralleled by enhanced remyelination, neuronal survival, and monoaminergic innervation, reduced astrogliosis, and activation of microglia. Together, these findings suggest that application of small organic compounds that bind to L1 and stimulate the beneficial homophilic L1 functions may prove to be a valuable addition to treatments of nervous system injuries. PMID:26253722

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

    PubMed

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

    2012-08-15

    Activated p53 is necessary for tumor suppression. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of functional p53. HIPK2 modulates wild-type p53 activity toward proapoptotic transcription and tumor suppression by the phosphorylation of serine 46. Knock-down of HIPK2 interferes with tumor suppression and sensitivity to chemotherapy. Combined administration of adriamycin and zinc restores activity of misfolded p53 and enables the induction of its proapoptotic and tumor suppressor functions in vitro and in vivo. We therefore looked for a cancer model where HIPK2 expression is low. MMTV-neu transgenic mice overexpressing HER2/neu, develop mammary tumors at puberty with a long latency, showing very low expression of HIPK2. Here we show that whereas these tumors are resistant to adriamycin treatment, a combination of adriamycin and zinc suppresses tumor growth in vivo in these mice, an effect evidenced by the histological features of the mammary tumors. The combined treatment of adriamycin and zinc also restores wild-type p53 conformation and induces proapoptotic transcription activity. These findings may open up new possibilities for the treatment of human cancers via the combination of zinc with chemotherapeutic agents, for a selected group of patients expressing low levels of HIPK2, with an intact p53. In addition, HIPK2 may serve as a new biomarker for tumor aggressiveness. PMID:21932419

  3. 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

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

    NASA Astrophysics Data System (ADS)

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

    1985-04-01

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

  5. In vivo imaging of the integration and function of nigral grafts in clinical trials.

    PubMed

    Politis, Marios; Piccini, Paola

    2012-01-01

    In vivo functional imaging has provided objective evidence for the integration and function of nigral grafts in the brains of patients with Parkinson's disease. Clinical trials with the use of positron emission tomography have shown that transplants of human dopamine-rich fetal ventral mesencephalic tissue can survive, grow, and release dopamine providing motor symptom relief, and also that they can restore brain activation related to movement. Positron emission tomography has aided in the elucidation of the pathophysiology of serious adverse effects, so-called graft-induced dyskinesias. With the use of newly established radioligands, positron emission tomography and single-photon emission computed tomography could help to improve Parkinson's patient selection in future clinical trials by selecting those with better predicted outcomes. Moreover, positron emission tomography could help monitoring postoperational inflammatory processes around the grafted tissue and the effect of immunosuppression. Recent evidence from positron emission tomography has provided insight of how ongoing extrastriatal serotonergic denervation may have relevance to nonmotor symptoms in transplanted Parkinson's disease patients indicating new cell therapy targets for a more complete relief of symptoms. Functional and structural magnetic resonance imaging techniques could help to better assess the integration of nigral graft with the host brain by assessing the restoration of brain activation during movement and of functional and structural connectivity. This knowledge should lead to the development of new, optimized in vivo imaging protocols that could help to better schedule, monitor, and modify the clinical outcomes of future human trials assessing the efficacy of fetal or stem cell therapy in Parkinson's disease. PMID:23195420

  6. Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation.

    PubMed

    Gong, Qian; Hazen, Meredith; Marshall, Brett; Crowell, Susan R; Ou, Qinglin; Wong, Athena W; Phung, Wilson; Vernes, Jean-Michel; Meng, Y Gloria; Tejada, Max; Andersen, Dana; Kelley, Robert F

    2016-01-01

    For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. Since effector function(s), including antibody-dependent cell-mediated cytotoxicity (ADCC), require the Fc portion to be glycosylated, reduced ADCC activity antibodies can be obtained through aglycosylation of the human IgG1 isotype. An alternative is to switch to an IgG4 isotype in which the glycosylated antibody is known to have reduced effector function relative to glycosylated IgG1 antibody. ADCC activity of glycosylated IgG1 antibodies is sensitive to the fucosylation status of the Fc glycan, with both in vitro and in vivo ADCC activity increased upon fucose removal ("afucosylation"). The effect of afucosylation on activity of IgG4 antibodies is less well characterized, but it has been shown to increase the in vitro ADCC activity of an anti-CD20 antibody. Here, we show that both in vitro and in vivo activity of anti-CD20 IgG4 isotype antibodies is increased via afucosylation. Using blends of material made in Chinese hamster ovary (CHO) and Fut8KO-CHO cells, we show that ADCC activity of an IgG4 version of an anti-human CD20 antibody is directly proportional to the fucose content. In mice transgenic for human FcγRIIIa, afucosylation of an IgG4 anti-mouse CD20 antibody increases the B cell depletion activity to a level approaching that of the mIgG2a antibody. PMID:27216702

  7. Quantitative analysis of bone and soft tissue by micro-computed tomography: applications to ex vivo and in vivo studies

    PubMed Central

    Campbell, Graeme M; Sophocleous, Antonia

    2014-01-01

    Micro-computed tomography (micro-CT) is a high-resolution imaging modality that is capable of analysing bone structure with a voxel size on the order of 10 μm. With the development of in vivo micro-CT, where disease progression and treatment can be monitored in a living animal over a period of time, this modality has become a standard tool for preclinical assessment of bone architecture during disease progression and treatment. For meaningful comparison between micro-CT studies, it is essential that the same parameters for data acquisition and analysis methods be used. This protocol outlines the common procedures that are currently used for sample preparation, scanning, reconstruction and analysis in micro-CT studies. Scan and analysis methods for trabecular and cortical bone are covered for the femur, tibia, vertebra and the full neonate body of small rodents. The analysis procedures using the software provided by ScancoMedical and Bruker are discussed, and the routinely used bone architectural parameters are outlined. This protocol also provides a section dedicated to in vivo scanning and analysis, which covers the topics of anaesthesia, radiation dose and image registration. Because of the expanding research using micro-CT to study other skeletal sites, as well as soft tissues, we also provide a review of current techniques to examine the skull and mandible, adipose tissue, vasculature, tumour severity and cartilage. Lists of recommended further reading and literature references are included to provide the reader with more detail on the methods described. PMID:25184037

  8. 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…

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

    PubMed Central

    2013-01-01

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

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

    PubMed

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

    2015-11-01

    Different authors have modelled myofascial tissue connectivity over a distance using cadaveric models, but in vivo models are scarce. The aim of this study was to evaluate the relationship between pelvic motion and deep fascia displacement in the medial gastrocnemius (MG). Deep fascia displacement of the MG was evaluated through automatic tracking with an ultrasound. Angular variation of the pelvis was determined by 2D kinematic analysis. The average maximum fascia displacement and pelvic motion were 1.501 ± 0.78 mm and 6.55 ± 2.47 °, respectively. The result of a simple linear regression between fascia displacement and pelvic motion for three task executions by 17 individuals was r = 0.791 (P < 0.001). Moreover, hamstring flexibility was related to a lower anterior tilt of the pelvis (r = 0.544, P < 0.024) and a lower deep fascia displacement of the MG (r = 0.449, P < 0.042). These results support the concept of myofascial tissue connectivity over a distance in an in vivo model, reinforce the functional concept of force transmission through synergistic muscle groups, and grant new perspectives for the role of fasciae in restricting movement in remote zones. PMID:26467242

  11. Metabolic Flux and Compartmentation Analysis in the Brain In vivo

    PubMed Central

    Lanz, Bernard; Gruetter, Rolf; Duarte, João M. N.

    2013-01-01

    Through significant developments and progresses in the last two decades, in vivo localized nuclear magnetic resonance spectroscopy (MRS) became a method of choice to probe brain metabolic pathways in a non-invasive way. Beside the measurement of the total concentration of more than 20 metabolites, 1H MRS can be used to quantify the dynamics of substrate transport across the blood-brain barrier by varying the plasma substrate level. On the other hand, 13C MRS with the infusion of 13C-enriched substrates enables the characterization of brain oxidative metabolism and neurotransmission by incorporation of 13C in the different carbon positions of amino acid neurotransmitters. The quantitative determination of the biochemical reactions involved in these processes requires the use of appropriate metabolic models, whose level of details is strongly related to the amount of data accessible with in vivo MRS. In the present work, we present the different steps involved in the elaboration of a mathematical model of a given brain metabolic process and its application to the experimental data in order to extract quantitative brain metabolic rates. We review the recent advances in the localized measurement of brain glucose transport and compartmentalized brain energy metabolism, and how these reveal mechanistic details on glial support to glutamatergic and GABAergic neurons. PMID:24194729

  12. Characterization of the Structural and Functional Determinants of MANF/CDNF in Drosophila In Vivo Model

    PubMed Central

    Lindström, Riitta; Lindholm, Päivi; Kallijärvi, Jukka; Yu, Li-ying; Piepponen, T. Petteri; Arumäe, Urmas; Saarma, Mart; Heino, Tapio I.

    2013-01-01

    Mammalian MANF and CDNF proteins are evolutionarily conserved neurotrophic factors that can protect and repair mammalian dopaminergic neurons in vivo. In Drosophila, the sole MANF protein (DmManf) is needed for the maintenance of dopaminergic neurites and dopamine levels. Although both secreted and intracellular roles for MANF and CDNF have been demonstrated, very little is known about the molecular mechanism of their action. Here, by using a transgenic rescue approach in the DmManf mutant background we show that only full-length MANF containing both the amino-terminal saposin-like and carboxy-terminal SAP-domains can rescue the larval lethality of the DmManf mutant. Independent N- or C-terminal domains of MANF, even when co-expressed together, fail to rescue. Deleting the signal peptide or mutating the CXXC motif in the C-terminal domain destroys the activity of full-length DmManf. Positively charged surface amino acids and the C-terminal endoplasmic reticulum retention signal are necessary for rescue of DmManf mutant lethality when DmManf is expressed in a restricted pattern. Furthermore, rescue experiments with non-ubiquitous expression reveals functional differences between the C-terminal domain of human MANF and CDNF. Finally, DmManf and its C-terminal domain rescue mammalian sympathetic neurons from toxin-induced apoptosis in vitro demonstrating functional similarity of the mammalian and fly proteins. Our study offers further insights into the functional conservation between invertebrate and mammalian MANF/CDNF proteins and reveals the importance of the C-terminal domain for MANF activity in vivo. PMID:24019940

  13. Demonstration of an in vivo functional beta 3-adrenoceptor in man.

    PubMed

    Enocksson, S; Shimizu, M; Lönnqvist, F; Nordenström, J; Arner, P

    1995-05-01

    Although it is well established in several mammalian species that beta 3-adrenoceptors play a major role in regulating lipolysis and thermogenesis in adipose tissue, the functional existence and role of this receptor subtype in man has been controversial. We investigated whether the beta 3-adrenoceptor functionally co-exists with beta 1- and beta 2-adrenoceptors in vivo in human adipose tissue. Subcutaneous abdominal adipose tissue of healthy non-obese subjects was microdialyzed with equimolar concentrations of dobutamine (selective beta 1-adrenoceptor agonist), terbutaline (selective beta 2-adrenoceptor agonist), or CGP 12177 (selective beta 3-adrenoceptor agonist). All three agents caused a rapid, sustained, concentration-dependent and significant elevation of the glycerol level in the microdialysate (lipolysis index). However, only terbutaline stimulated the nutritive blood flow in adipose tissue, as measured by an ethanol escape technique. Dobutamine and CGP 12177 was equally effective in elevating the glycerol level (maximum effect 150% above baseline). Terbutaline was significantly more effective than the other two beta-agonists (maximum effect 200% above baseline). When adipose tissue was pretreated with the beta 1/beta 2-selective adrenoceptor blocker propranolol the glycerol increasing effect of dobutamine or terbutaline was inhibited by 80-85% but the glycerol response to CGP 12177 was not influenced. It is concluded that a functional beta 3-adrenoceptor is present in vivo in man. It co-exists with beta 1- and beta 2-adrenoceptors in adipose tissue and may therefore play a role in lipolysis regulation. It appears, however, that the beta 2-adrenoceptor is the most important beta-adrenoceptor subtype for the mobilization of lipids from abdominal subcutaneous adipose tissue because of its concomitant stimulatory effect on lipolysis and blood flow. PMID:7738189

  14. Functional Analysis of Transcription Factors in Arabidopsis

    PubMed Central

    Mitsuda, Nobutaka; Ohme-Takagi, Masaru

    2009-01-01

    Transcription factors (TFs) regulate the expression of genes at the transcriptional level. Modification of TF activity dynamically alters the transcriptome, which leads to metabolic and phenotypic changes. Thus, functional analysis of TFs using ‘omics-based’ methodologies is one of the most important areas of the post-genome era. In this mini-review, we present an overview of Arabidopsis TFs and introduce strategies for the functional analysis of plant TFs, which include both traditional and recently developed technologies. These strategies can be assigned to five categories: bioinformatic analysis; analysis of molecular function; expression analysis; phenotype analysis; and network analysis for the description of entire transcriptional regulatory networks. PMID:19478073

  15. Single Amino Acid Mutations in Drosophila Fascin Disrupt Actin Bundling Function in Vivo

    PubMed Central

    Cant, K.; Cooley, L.

    1996-01-01

    Fascins bundle actin filaments into large, tightly packed hexagonal arrays that support diverse cellular processes including microvillar projections and filopodial extensions. In Drosophila, fascin is encoded by the singed locus. Severe singed mutants have gnarled bristles and are female sterile due to a defect in rapid cytoplasm transport during oogenesis. In this paper, we report the results of a large EMS mutagenesis screen to generate new singed alleles. A mutation that changes glycine 409 to glutamic acid results in partial inactivation of fascin in vivo, singed(G409E) mutants have kinked bristles and are fertile with a mild nurse cell cytoplasm transport defect. This mutation is in a small conserved domain near the C-terminus of fascin. A mutation that changes serine 289 to asparagine almost completely inactivates fascin in vivo, singed(S289N) mutants have gnarled bristles and are sterile due to a severe defect in nurse cell cytoplasm transport caused by the absence of nurse cell cytoplasmic actin bundles. A subsequent EMS mutagenesis screen for dominant suppressors of singed(S289N) sterility revealed an intragenic suppressor mutation that changes serine 251 to phenylalanine and restores much of fascin's function. These two mutations, S289N and S251F, draw attention to a central domain in fascin. PMID:8722779

  16. 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

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

    SciTech Connect

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

    2000-11-15

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

  18. 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

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

    PubMed Central

    Kaiser, Nicholas J; Coulombe, Kareen L K

    2015-01-01

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

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

    PubMed Central

    Soda, Naoki; Fujihashi, Yuichiro; Aoki, Takaaki

    2016-01-01

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

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

    PubMed Central

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

    2009-01-01

    Uterine bacterial infection after parturition causes endometritis, perturbs ovarian function and leads to infertility in cattle. Although endometritis is caused by mixed infections, endometrial pathology is associated with the presence of Arcanobacterium pyogenes. The aims of the present study were to determine the effects of A. pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo. Heat-killed A. pyogenes did not affect the production of prostaglandin F2α (PGF) or prostaglandin E2 (PGE) from endometrial explants, or purified populations of endometrial epithelial or stromal cells. However, the explants produced more PGF and PGE than controls when treated with a bacteria-free filtrate (BFF) cultured from A. pyogenes. Similarly, BFF stimulated PGF and PGE production by epithelial and stromal cells, respectively. So, BFF or control PBS was infused into the uterus of heifers (n = 7 per group) for 8 days, starting the day after estrus. Emergence of the follicle wave, dominant follicle or corpus luteum diameter, and peripheral plasma FSH, LH, estradiol, progesterone, PGFM, or acute phase protein concentrations were unaffected by the BFF infusion. In the live animal it is likely that the intact uterine mucosa limits the exposure of the endometrial cells to the exotoxin of A. pyogenes, whereas the cells are readily exposed to the toxin in vitro. PMID:17825901

  2. Analysis of the kinesin superfamily: insights into structure and function.

    PubMed

    Miki, Harukata; Okada, Yasushi; Hirokawa, Nobutaka

    2005-09-01

    Kinesin superfamily proteins (KIFs) are key players or 'hub' proteins in the intracellular transport system, which is essential for cellular function and morphology. The KIF superfamily is also the first large protein family in mammals whose constituents have been completely identified and confirmed both in silico and in vivo. Numerous studies have revealed the structures and functions of individual family members; however, the relationships between members or a perspective of the whole superfamily structure until recently remained elusive. Here, we present a comprehensive summary based on a large, systematic phylogenetic analysis of the kinesin superfamily. All available sequences in public databases, including genomic information from all model organisms, were analyzed to yield the most complete phylogenetic kinesin tree thus far, comprising 14 families. This comprehensive classification builds on the recently proposed standardized nomenclature for kinesins and allows systematic analysis of the structural and functional relationships within the kinesin superfamily. PMID:16084724

  3. A non-linear mathematical model for the in vivo evaluation of the RES phagocytic function.

    PubMed

    Bondareva, I B; Parfenov, A S

    1995-01-01

    A new non-linear mathematical model was constructed in order to perform in vivo quantification of the RES phagocytic function. This method is based on the same technical facilities as used for the routine liver-spleen scintigraphy with radiocolloids [1, 2]. But kinetic modeling of dynamic Tc-99m-sulfur colloid data produced estimations of the functional RE-parameters: the clearance rate of the colloidal particles, the rate of phagocytosis, and the RES functional volume, which can not be obtained by classical approaches. This non-linear model was designed on the basis of the principal characteristics of particulate material interaction with macrophages (attachment, phagocytosis, digestion) [3, 4, 5]. The theoretically examined behavior of this in vivo mathematical model corresponds with the experimental behavior of the RES. The mathematical expression of the dynamics is the system of non-linear differential equations with constant coefficients that have no analytical solution. Fitting of the normalized heart blood time-activity curve was obtained to identify the unknown model parameters via non-linear regression. For this purpose general interactive PASCAL procedure IDPAR for a PDP-11/34 computer was used (an IBM PC version is also available). Two to three iterations were needed to estimate the set of unknown parameters for any patient study (1-1.5 min). A very good fitting was obtained between experimental and model curves in every case of different pathologies (error of the approximation is about 2-3%). Studies were performed using an in vivo bolus injection of 3.6 mg/80 kg commercially available colloid KOREN labeled with 3m-Ci 99m-Tc (analog of TCK-1). Our method was used to determine the RES functional parameters for patient groups with different levels of the RES dysfunction. Obtained results illustrate the possibilities of our technique to quantitatively estimate not only great pathology (portal cirrhosis), but also small changes of the RE-function (case of

  4. Rapid Biocompatibility Analysis of Materials via In Vivo Fluorescence Imaging of Mouse Models

    PubMed Central

    Bratlie, Kaitlin M.; Dang, Tram T.; Lyle, Stephen; Nahrendorf, Matthias; Weissleder, Ralph; Langer, Robert; Anderson, Daniel G.

    2010-01-01

    Background Many materials are unsuitable for medical use because of poor biocompatibility. Recently, advances in the high throughput synthesis of biomaterials has significantly increased the number of potential biomaterials, however current biocompatibility analysis methods are slow and require histological analysis. Methodology/Principal Findings Here we develop rapid, non-invasive methods for in vivo quantification of the inflammatory response to implanted biomaterials. Materials were placed subcutaneously in an array format and monitored for host responses as per ISO 10993-6: 2001. Host cell activity in response to these materials was imaged kinetically, in vivo using fluorescent whole animal imaging. Data captured using whole animal imaging displayed similar temporal trends in cellular recruitment of phagocytes to the biomaterials compared to histological analysis. Conclusions/Significance Histological analysis similarity validates this technique as a novel, rapid approach for screening biocompatibility of implanted materials. Through this technique there exists the possibility to rapidly screen large libraries of polymers in vivo. PMID:20386609

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

    PubMed

    Epifanio, Irene; Ventura-Campos, Noelia

    2014-02-28

    The hippocampus is one of the first affected regions in Alzheimer's disease. The left hippocampi of control subjects, patients with mild cognitive impairment and patients with Alzheimer's disease are represented by spherical harmonics. Functional data analysis is used in the hippocampal shape analysis. Functional principal component analysis and functional independent component analysis are defined for multivariate functions with two arguments. A functional linear discriminant function is also defined. Comparisons with other approaches are carried out. Our functional approach gives promising results, especially in shape classification. PMID:24105806

  6. In vivo Analysis of White Adipose Tissue in Zebrafish

    PubMed Central

    Minchin, James E.N.; Rawls, John F.

    2016-01-01

    White adipose tissue (WAT) is the major site of energy storage in bony vertebrates, and also serves central roles in the endocrine regulation of energy balance. The cellular and molecular mechanisms underlying WAT development and physiology are not well understood. This is due in part to difficulties associated with imaging adipose tissues in mammalian model systems, especially during early life stages. The zebrafish (Danio rerio) has recently emerged as a new model system for adipose tissue research, in which WAT can be imaged in a transparent living vertebrate at all life stages. Here we present detailed methods for labeling adipocytes in live zebrafish using fluorescent lipophilic dyes, and for in vivo microscopy of zebrafish WAT. PMID:21951526

  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. 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.

  9. Formulation/Preparation of Functionalized Nanoparticles for In Vivo Targeted Drug Delivery

    NASA Astrophysics Data System (ADS)

    Gu, Frank; Langer, Robert; Farokhzad, Omid C.

    Targeted cancer therapy allows the delivery of therapeutic agents to cancer cells without incurring undesirable side effects on the neighboring healthy tissues. Over the past decade, there has been an increasing interest in the development of advanced cancer therapeutics using targeted nanoparticles. Here we describe the preparation of drug-encapsulated nanoparticles formulated with biocompatible and biodegradable poly( d, l-lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG) copolymer and surface functionalized with the A10 2-fluoropyrimidine ribonucleic acid aptamers that recognize the extracellular domain of prostate-specific membrane antigen (PSMA), a well-characterized antigen expressed on the surface of prostate cancer cells. We show that the self-assembled nanoparticles can selectively bind to PSMA-targeted prostate cancer cells in vitro and in vivo. This formulation method may contribute to the development of highly selective and effective cancer therapeutic and diagnostic devices.

  10. A comprehensive analysis of microRNA expression during human keratinocyte differentiation in vitro and in vivo.

    PubMed

    Hildebrand, Janosch; Rütze, Martin; Walz, Nicole; Gallinat, Stefan; Wenck, Horst; Deppert, Wolfgang; Grundhoff, Adam; Knott, Anja

    2011-01-01

    Here, we report a comprehensive investigation of changes in microRNA (miRNA) expression profiles on human keratinocyte (HK) differentiation in vitro and in vivo. We have monitored expression patterns of 377 miRNAs during calcium-induced differentiation of primary HKs, and have compared these patterns with miRNA expression profiles of epidermal stem cells, transient amplifying cells, and terminally differentiated HKs from human skin. Apart from the previously described miR-203, we found an additional nine miRNAs (miR-23b, miR-95, miR-210, miR-224, miR-26a, miR-200a, miR-27b, miR-328, and miR-376a) that are associated with HK differentiation in vitro and in vivo. In situ hybridization experiments confirmed miR-23b as a marker of HK differentiation in vivo. Additionally, gene ontology analysis and functional validation of predicted miRNA targets using 3'-untranslated region-luciferase assays suggest that multiple miRNAs that are upregulated on HK differentiation cooperate to regulate gene expression during skin development. Our results thus provide the basis for further analysis of miRNA functions during epidermal differentiation. PMID:20827281

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

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

    2010-09-01

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

  13. 40 CFR 798.5385 - In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the... cytogenetics tests: Chromosomal analysis. 798.5385 Section 798.5385 Protection of Environment ENVIRONMENTAL... Genetic Toxicity § 798.5385 In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis....

  14. Rapid Immunomagnetic Negative Enrichment of Neutrophil Granulocytes from Murine Bone Marrow for Functional Studies In Vitro and In Vivo

    PubMed Central

    Hasenberg, Mike; Köhler, Anja; Bonifatius, Susanne; Borucki, Katrin; Riek-Burchardt, Monika; Achilles, Julia; Männ, Linda; Baumgart, Kathleen; Schraven, Burkhart; Gunzer, Matthias

    2011-01-01

    Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*106 PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo. PMID:21383835

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2006-10-01

    Naturally occurring CD4+ regulatory T cells (T(R)) that express CD25 and the transcription factor FoxP3 play a key role in immune homeostasis, preventing immune pathological responses to self and foreign Ags. CTLA-4 is expressed by a high percentage of these cells, and is often considered as a marker for T(R) in experimental and clinical analysis. However, it has not yet been proven that CTLA-4 has a direct role in T(R) function. In this study, using a T cell-mediated colitis model, we demonstrate that anti-CTLA-4 mAb treatment inhibits T(R) function in vivo via direct effects on CTLA-4-expressing T(R), and not via hyperactivation of colitogenic effector T cells. Although anti-CTLA-4 mAb treatment completely inhibits T(R) function, it does not reduce T(R) numbers or their homing to the GALT, suggesting the Ab mediates its function by blockade of a signal required for T(R) activity. In contrast to the striking effect of the Ab, CTLA-4-deficient mice can produce functional T(R), suggesting that under some circumstances other immune regulatory mechanisms, including the production of IL-10, are able to compensate for the loss of the CTLA-4-mediated pathway. This study provides direct evidence that CTLA-4 has a specific, nonredundant role in the function of normal T(R). This role has to be taken into account when targeting CTLA-4 for therapeutic purposes, as such a strategy will not only boost effector T cell responses, but might also break T(R)-mediated self-tolerance. PMID:16982872

  17. HIV Type 1 Infection Up-Regulates TLR2 and TLR4 Expression and Function in Vivo and in Vitro

    PubMed Central

    Hernández, Juan C.; Stevenson, Mario; Latz, Eicke

    2012-01-01

    Abstract Toll-like receptors (TLRs) play a critical role in innate immunity against pathogens. Their stimulation induces the activation of NF-κB, an important inducer of HIV-1 replication. In recent years, an increasing number of studies using several cells types from HIV-infected patients indicate that TLRs play a key role in regulating the expression of proinflammatory cytokines and viral pathogenesis. In the present study, the effect of HIV-1 stimulation of monocyte-derived macrophage (MDM) and peripheral blood mononuclear cell (PBMC) subpopulations from healthy donors on the expression and functions of TLR2 and TLR4 was examined. In addition, and to complete the in vitro study, the expression pattern of TLR2 and TLR4 in 49 HIV-1-infected patients, classified according to viral load and the use of HAART, was determined and compared with 25 healthy subjects. An increase of TLR expression and production of proinflammatory cytokines were observed in MDMs and PBMCs infected with HIV-1 in vitro and in response to TLR stimulation, compared to the mock. In addition, an association between TLR expression and up-regulation of CD80 in plasmacytoid dendritic cells (pDCs) was observed. The ex vivo analysis indicated increased expression of TLR2 and TLR4 in myeloid dendritic cells (mDCs), but only of TLR2 in monocytes obtained from HIV-1-infected patients, compared to healthy subjects. Remarkably, the expression was higher in cells from patients who do not use HAART. In monocytes, there was a positive correlation between both TLRs and viral load, but not CD4+ T cell numbers. Together, our in vitro and ex vivo results suggest that TLR expression and function can be up-regulated in response to HIV-1 infection and could affect the inflammatory response. We propose that modulation of TLRs represents a mechanism to promote HIV-1 replication or AIDS progression in HIV-1-infected patients. PMID:22280204

  18. 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 μm 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

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

    PubMed

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

    2011-08-01

    Abuse of the powerfully addictive psychostimulant, methamphetamine, occurs worldwide. Recent studies have suggested that methamphetamine-induced dopaminergic neurotoxicity is related to oxidative stress. In response to nerve activation, the mitochondrial respiratory chain is rapidly activated. The enhancement of mitochondrial respiratory chain activation may induce oxidative stress in the brain. However, there is little experimental evidence regarding the mitochondrial function after methamphetamine administration in vivo. Here, we evaluated whether a single administration of methamphetamine induces ATP consumption and overactivation of mitochondria. We measured mitochondrial function in two different ways: by monitoring oxygen partial pressure using an oxygen-selective electrode, and by imaging of redox reactions using a nitroxyl radical (i.e., nitroxide) coupled with Overhauser-enhanced magnetic resonance imaging (OMRI). A single administration of methamphetamine to Wistar rats induced dopaminergic nerve activation, ATP consumption and an increase in mitochondrial respiratory chain function in both the striatum and cortex. Furthermore, antioxidant TEMPOL prevented the increase in mitochondrial oxidative damage and methamphetamine-induced sensitization. These findings suggest that energy-supplying reactions after dopaminergic nerve activation are associated with oxidative stress in both the striatum and cortex, leading to abnormal behavior. PMID:21624473

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

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

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

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

    PubMed 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  4. Structure predicts function: combining non-invasive electrophysiology with in-vivo histology.

    PubMed

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

    2015-03-01

    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 800μm 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

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

    PubMed Central

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

    2016-01-01

    Optical Coherence Tomography (OCT) enables real-time imaging of living tissues at cell-scale resolution over millimeters in three dimensions. Despite these advantages, functional biological studies with OCT have been limited by a lack of exogenous contrast agents that can be distinguished from tissue. Here we report an approach to functional OCT imaging that implements custom algorithms to spectrally identify unique contrast agents: large gold nanorods (LGNRs). LGNRs exhibit 110-fold greater spectral signal per particle than conventional GNRs, which enables detection of individual LGNRs in water and concentrations as low as 250 pM in the circulation of living mice. This translates to ~40 particles per imaging voxel in vivo. Unlike previous implementations of OCT spectral detection, the methods described herein adaptively compensate for depth and processing artifacts on a per sample basis. Collectively, these methods enable high-quality noninvasive contrast-enhanced imaging of OCT in living subjects, including detection of tumor microvasculature at twice the depth achievable with conventional OCT. Additionally, multiplexed detection of spectrally-distinct LGNRs was demonstrated to observe discrete patterns of lymphatic drainage and identify individual lymphangions and lymphatic valve functional states. These capabilities provide a powerful platform for molecular imaging and characterization of tissue noninvasively at cellular resolution, called MOZART. PMID:26987475

  6. 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

  7. Fiberless multicolor neural optoelectrode for in vivo circuit analysis.

    PubMed

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

    2016-01-01

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

  8. In Vivo Analysis of Trapeziometacarpal Joint Kinematics during Pinch Tasks

    PubMed Central

    Chen, Guan-Po; Jou, I-Ming; Goryacheva, Irina G.; Dosaev, Marat Z.; Su, Fong-Chin

    2014-01-01

    This study investigated how the posture of the thumb while performing common pinch movements and the levels of pinch force applied by the thumb affect the arthrokinematics of the trapeziometacarpal joint in vivo. Fifteen subjects performed the pinch tasks at the distal phalange (DP), proximal interphalangeal (PIP) joint, and metacarpophalangeal (MP) joint of the index finger with 0%, 50%, and 80% of maximal pinch forces by a single-axis load cell. 3D images of the thumb were obtained using the computed tomography. The results show that the reference points moved from the central region to the dorsal-radial region when changing from pinching the DP to the MP joint without pinching force being applied. Pinching with 80% of the maximum pinching force resulted in reference points being the closest to the volar-ulnar direction. Significant differences were seen between 0% and 50% of maximum pinch force, as well as between 0% and 80%, when pinching the MP joint in the distal-proximal direction. The effects of posture of the thumb and applied pinch force on the arthrokinematics of the joint were investigated with a 3D model of the trapeziometacarpal joint. Pinching with more than 50% of maximum pinch force might subject this joint to extreme displacement. PMID:24683540

  9. Fiberless multicolor neural optoelectrode for in vivo circuit analysis

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  10. 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.

  11. In vivo analysis of trapeziometacarpal joint kinematics during pinch tasks.

    PubMed

    Kuo, Li-Chieh; Lin, Chien-Ju; Chen, Guan-Po; Jou, I-Ming; Wang, Chien-Kuo; Goryacheva, Irina G; Dosaev, Marat Z; Su, Fong-Chin

    2014-01-01

    This study investigated how the posture of the thumb while performing common pinch movements and the levels of pinch force applied by the thumb affect the arthrokinematics of the trapeziometacarpal joint in vivo. Fifteen subjects performed the pinch tasks at the distal phalange (DP), proximal interphalangeal (PIP) joint, and metacarpophalangeal (MP) joint of the index finger with 0%, 50%, and 80% of maximal pinch forces by a single-axis load cell. 3D images of the thumb were obtained using the computed tomography. The results show that the reference points moved from the central region to the dorsal-radial region when changing from pinching the DP to the MP joint without pinching force being applied. Pinching with 80% of the maximum pinching force resulted in reference points being the closest to the volar-ulnar direction. Significant differences were seen between 0% and 50% of maximum pinch force, as well as between 0% and 80%, when pinching the MP joint in the distal-proximal direction. The effects of posture of the thumb and applied pinch force on the arthrokinematics of the joint were investigated with a 3D model of the trapeziometacarpal joint. Pinching with more than 50% of maximum pinch force might subject this joint to extreme displacement. PMID:24683540

  12. Fiberless multicolor neural optoelectrode for in vivo circuit analysis

    PubMed Central

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

    2016-01-01

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

  13. 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.

  14. Structure and function analysis of protein–nucleic acid complexes

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  15. 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.

  16. Relationship between in vitro sperm functional tests and in vivo fertility of rams following cervical artificial insemination of ewes with frozen-thawed semen.

    PubMed

    O' Meara, C M; Hanrahan, J P; Prathalingam, N S; Owen, J S; Donovan, A; Fair, S; Ward, F; Wade, M; Evans, A C O; Lonergan, P

    2008-03-01

    Several procedures have been proposed to assess structural and functional characteristics of cryopreserved ram semen but none so far have yielded consistent relationships with in vivo fertility. The objectives of this study were to evaluate several sperm function tests as potential markers of in vivo ram fertility (determined by pregnancy rate in ewes) using frozen-thawed semen. In experiment 1, frozen-thawed straws (n=3 per ram) of semen from three high and three low fertility rams were assessed using fluorescent microscopy for (1) progressive motility, (2) viability and, (3) acrosomal status. In experiment 2, frozen-thawed straws (n=3 per ram) of semen from 18 rams of known fertility were analysed using either computer-assisted sperm analysis (CASA) for eight motion characteristics or flow cytometric staining for: (1) viability and acrosomal status, (2) plasma membrane status and capacitation-like changes, and (3) live cells following an osmotic resistance test (ORT). In experiment 3, platelet-activating factor (PAF) was isolated from straws (n=2 per ram) of semen using high-pressure liquid chromatography (HPLC) and quantified using HPLC-tandem mass spectrometry for 18 rams. In experiment 1, no association was found between motility, viability (% live) or acrosomal status (% damaged, % intact and % reacted) and in vivo fertility. In experiment 2, no correlation was found between motility (CASA), viability (% live), acrosomal status (% live, % live intact and % reacted), capacitation status (% capacitated, % non-capacitated), plasma membrane stability (% dead) and % live cells following ORT and ram in vivo fertility. In experiment 3, there was no relationship between PAF content in spermatozoa and ram fertility. In conclusion, we were unable to relate the in vivo fertility of rams with in vitro functional tests of their frozen-thawed semen and suggest that the fertility of a given semen sample cannot easily be quantified using available in vitro tests. PMID

  17. Flexible polyimide microelectrode array for in vivo recordings and current source density analysis.

    PubMed

    Cheung, Karen C; Renaud, Philippe; Tanila, Heikki; Djupsund, Kaj

    2007-03-15

    This work presents implantable, flexible polymer-based probes with embedded microelectrodes for acute and chronic neural recordings in vivo, as tested on rodents. Acute recordings using this array were done in mice under urethane anesthesia and compared to those made using silicon-based probes manufactured at the Center for Neural Communication Technology, University of Michigan. The two electrode arrays yielded similar results. Recordings with chronically implanted polymer-based electrodes were performed for 60 days post-surgically in awake, behaving rats. The microelectrodes were used to monitor local field potentials and capture laminar differences in function of cortex and hippocampus, and produced response waveforms of undiminished amplitude and signal-to-noise ratios 8 weeks after chronic implantation. The polymer-based electrodes could also be connected to a lesion current to mark specific locations in the tissue. Current source density (CSD) analysis from the recordings depicted a source - sink-composition. Tissue response was assessed 8 weeks after insertion by immunochemical labeling with glial fibrillary acidic protein (GFAP) to identify astrocytes, and histological analysis showed minimal tissue reaction to the implanted structures. PMID:17027251

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

    PubMed

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

    2009-01-01

    Polymer chemistry offers the possibility of synthesizing multifunctional nanoparticles which incorporate moieties that enhance diagnostic and therapeutic targeting of cargo delivery to the lung. However, since rules for predicting particle behavior following modification are not well-defined, it is essential that probes for tracking fate in vivo are also included. Accordingly, we designed polyacrylamide-based hydrogel particles of differing sizes, functionalized with a nona-arginine cell-penetrating peptide (Arg(9)), and labeled with imaging components to assess lung retention and cellular uptake after intratracheal administration. Radiolabeled microparticles (1-5 microm diameter) and nanoparticles (20-40 nm diameter) without and with Arg(9) showed diffuse airspace distribution by positron emission tomography imaging. Biodistribution studies revealed that particle clearance and extrapulmonary distribution was, in part, size dependent. Microparticles were rapidly cleared by mucociliary routes but, unexpectedly, also through the circulation. In contrast, nanoparticles had prolonged lung retention enhanced by Arg(9) and were significantly restricted to the lung. For all particle types, uptake was predominant in alveolar macrophages and, to a lesser extent, lung epithelial cells. In general, particles did not induce local inflammatory responses, with the exception of microparticles bearing Arg(9). Whereas microparticles may be advantageous for short-term applications, nanosized particles constitute an efficient high-retention and non-inflammatory vehicle for the delivery of diagnostic imaging agents and therapeutics to lung airspaces and alveolar macrophages that can be enhanced by Arg(9). Importantly, our results show that minor particle modifications may significantly impact in vivo behavior within the complex environments of the lung, underscoring the need for animal modeling. PMID:19852512

  19. Molecular organization and in vivo function of the cytoskeleton of amphibian erythrocytes.

    PubMed

    Lee, Kyeng Gea; Kerr, Louis M; Cohen, William D

    2007-08-01

    One prominent cytoskeletal feature of non-mammalian vertebrate erythrocytes is the marginal band (MB), composed of microtubules. However, there have been several reports of MB-associated F-actin. We have further investigated the function of MB-associated F-actin, using newt erythrocytes having large, thick MBs. Confocal microscopy revealed a distinctive band of F-actin colocalizing point- by-point with MB microtubules. Furthermore, the F-actin band was present in isolated elliptical MBs, but absent in membrane skeletons lacking MBs. F-actin depolymerizing agents did not affect F-actin band integrity in isolated MBs, indicating its non-dynamic state. However, exposure to elastase resulted in F-actin removal and MB circularization. These results provide evidence of a strong association of F-actin with MB microtubules in mature ellipsoidal erythrocytes. To assess the true extent of mechanical stress on the cytoskeleton, erythrocytes were observed by video microscopy during flow in vivo. Moving with long axis parallel to flow direction, cells underwent reversible shape distortion as they collided vigorously with other erythrocytes and vessel walls. In addition, cells twisted into figure-8 shapes, a cytoskeletal property that may provide physiological advantages during flow. Our results, together with those of others, yield a consistent picture in which developing erythrocytes undergo transition from spheroids to immature discoids to mature ellipsoids. The causal step in discoid formation is biogenesis of circular MBs with sufficient flexural rigidity to determine cell shape. F-actin binding to MB microtubules then creates a composite system, enhancing flexural rigidity to produce and maintain ellipsoidal shape during the physical challenges of blood flow in vivo. PMID:17508361

  20. 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

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

    PubMed

    Dehghani, Mehrnoush; Lasko, Paul

    2015-01-01

    The maternally expressed Drosophila melanogaster DEAD-box helicase Vasa (Vas) is necessary for many cellular and developmental processes, including specification of primordial germ cells (pole cells), posterior patterning of the embryo, piRNA-mediated repression of transposon-encoded mRNAs, translational activation of gurken (grk) mRNA, and completion of oogenesis itself. Vas protein accumulates in the perinuclear nuage in nurse cells soon after their specification, and then at stage 10 Vas translocates to the posterior pole plasm of the oocyte. We produced a series of transgenic constructs encoding eGFP-Vas proteins carrying mutations affecting different regions of the protein, and analyzed in vivo which Vas functions each could support. We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification. One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases. Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression. We conclude from these experiments that Vas, a multifunctional protein, uses different domains and different molecular associations to carry out its various cellular and developmental roles. PMID:25795910

  2. Function and viability of human islets encapsulated in alginate sheets: in vitro and in vivo culture.

    PubMed

    Lamb, M; Storrs, R; Li, S; Liang, O; Laugenour, K; Dorian, R; Chapman, D; Ichii, H; Imagawa, D; Foster, C; King, S; Lakey, J R T

    2011-11-01

    Islet encapsulation offers an immune system barrier for islet transplantation, and encapsulation within an alginate sheetlike structure offers the ability to be retrievable after transplanted. This study aims to show that human islets encapsulated into islet sheets remain functional and viable after 8 weeks in culture or when transplanted into the subcutaneous space of rats. Human islets were isolated from cadaveric organs. Dissociation and purification were done using enzymatic digestion and a continuous Ficoll-UWD gradient. Purified human islets were encapsulated in alginate sheets. Human Islet sheets were either kept in culture, at 37°C and 5% CO(2), or transplanted subcutaneously into Lewis rats. After 1, 2, 4, and 8 weeks, the human islet sheets were retrieved from the rats and assessed. The viability of the sheets was measured using fluorescein diacetate (FDA)/propidium iodide (PI), and function was measured through glucose-stimulated insulin release, in which the sheets were incubated for an hour in low-glucose concentration (2.8 mmol/L) and then high (28 mmol/L), then high (28 mmol/L) plus 3-isobutyl-1-methylxanthine (50 μm). Human islet sheets remained both viable, above 70%, and functional, with a stimulation index (insulin secretion in high glucose divided by insulin secretion in low glucose) above 1.5, over 8 weeks of culture or subcutaneous transplantation. Islet transplantation continues to make advances in the treatment of type 1 diabetes. These preliminary results suggest that encapsulated islets sheets can survive and maintain islet viability and function in vivo, within the subcutaneous region. PMID:22099772

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

    PubMed Central

    Dehghani, Mehrnoush; Lasko, Paul

    2015-01-01

    The maternally expressed Drosophila melanogaster DEAD-box helicase Vasa (Vas) is necessary for many cellular and developmental processes, including specification of primordial germ cells (pole cells), posterior patterning of the embryo, piRNA-mediated repression of transposon-encoded mRNAs, translational activation of gurken (grk) mRNA, and completion of oogenesis itself. Vas protein accumulates in the perinuclear nuage in nurse cells soon after their specification, and then at stage 10 Vas translocates to the posterior pole plasm of the oocyte. We produced a series of transgenic constructs encoding eGFP-Vas proteins carrying mutations affecting different regions of the protein, and analyzed in vivo which Vas functions each could support. We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification. One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases. Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression. We conclude from these experiments that Vas, a multifunctional protein, uses different domains and different molecular associations to carry out its various cellular and developmental roles. PMID:25795910

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

    PubMed

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

    2016-01-01

    Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca(2+)-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and - with the addition of its cofactor coelenterazine - emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca(2+)-transients and Ca(2+)-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca(2+)-activity within the mushroom bodies, a structure central to learning and memory in the fly brain. PMID:26779599

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

    PubMed Central

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

    2013-01-01

    The primacy and power of RNA in governing many processes of life has begun to be more fully appreciated in both the discovery and inventive sciences. A variety of RNA interactions regulate gene expression, and structural self-assembly underlies many of these processes. The understanding sparked by these discoveries has inspired and informed the engineering of novel RNA structures, control elements, and genetic circuits in cells. Many of these engineered systems are built up fundamentally from RNA–RNA interactions, often combining modular, rational design with functional selection and screening. It is therefore useful to review the particular class of RNA-based regulatory mechanisms that rely on RNA self-assembly either through homomeric (self–self) or heteromeric (self–nonself) RNA–RNA interactions. Structures and sequence elements within individual RNAs create a basis for the pairing interactions, and in some instances can even lead to the formation of RNA polymers. Example systems of dimers, multimers, and polymers are reviewed in this article in the context of natural systems, wherein the function and impact of self-assemblies are understood. Following this, a brief overview is presented of specific engineered RNA self-assembly systems implemented in vivo, with lessons learned from both discovery and engineering approaches to RNA–RNA self-assembly. PMID:23914307

  6. Effects of altered ventilatory patterns of rabbit pulmonary endothelial angiotensin converting enzyme function, in vivo

    SciTech Connect

    Toivonen, H.J.; Catravas, J.D.

    1986-03-01

    Because alveolar pressure can influence pulmonary blood flow, volume and surface area, the authors have studied the effects of airway pressure on endothelial angiotensin converting enzyme (ACE) function in rabbit lungs in vivo, utilizing indicator dilution techniques with /sup 3/H-Benzoyl-Phe-Ala-Pro (BPAP) as substate. Static inclation of the lungs to a pressure of 0 or 5 mmHg did not change percent transpulmonary metabolism and Amax/Km ratio in comparison to control measurements during conventional mechanical ventilation. When the inflation pressure was increased to 10 mmHg, percent metabolism of /sup 3/H-BPAP remained unaltered but Amax/Km decreased over 40% from control. This decrease was in close relation to the reduction in pulmonary blood flow. Addition of 5 cm H/sub 2/O positive end-expiratory pressure (PEEP) to the mechanical ventilation also decreased Amax/Km values and pulmonary blood flow but did not influence percent metabolism of /sup 3/H-BPAP. These results suggest that the detected alterations in ACE kinetics were more likely due to hemodynamic changes than enzyme dysfunction. The authors propose that high static alveolar pressures as well as PEEP did not affect angiotensin converting enzyme function, but reduced the fraction of perfused microvessels reflected in changes in Amax/Km ratios.

  7. Bridging the gap: functional healing of embryonic small intestine ex vivo.

    PubMed

    Coletta, Riccardo; Roberts, Neil A; Oltrabella, Francesca; Khalil, Basem A; Morabito, Antonino; Woolf, Adrian S

    2016-02-01

    The ability to grow embryonic organs ex vivo provides an opportunity to follow their differentiation in a controlled environment, with resulting insights into normal development. Additionally, similar strategies can be used to assess effects on organogenesis of physical and chemical manipulations. This study aimed to create an organ culture model with which to test physical manipulations to enhance healing of gut segments, thus generating a single functional organ. Embryonic mouse jejunum was isolated and cut into 2-3 mm tubes, which were placed in pairs, separated by a small gap, on semi-permeable supports. Each pair was linked by a nylon suture threaded through their lumens. After 3 days in organ culture fed by defined serum-free media, the rudiments differentiated to form tubes of smooth muscle surrounding a core of rudimentary villi. Of 34 such pairs, 74% had touching and well aligned proximate ends. Of these joined structures, 80% (59% of the total pairs) had a continuous lumen, as assessed by observing the trajectories of fluorescent dextrans injected into their distal ends. Fused organ pairs formed a single functional unit, as assessed by spontaneous contraction waves propagated along their lengths. In these healed intestines, peripherin(+) neurons formed a nexus in the zone of fusion, linking the rudiment pairs. In future, this system could be used to test whether growth factors enhance fusion. Such results should in turn inform the design of novel treatments for short bowel syndrome, a potentially fatal condition with a currently limited and imperfect range of therapies. PMID:26234729

  8. Truncated HP1 lacking a functional chromodomain induces heterochromatinization upon in vivo targeting.

    PubMed

    Brink, Maartje C; van der Velden, Yme; de Leeuw, Wim; Mateos-Langerak, Julio; Belmont, Andrew S; van Driel, Roel; Verschure, Pernette J

    2006-01-01

    Packaging of the eukaryotic genome into higher order chromatin structures is tightly related to gene expression. Pericentromeric heterochromatin is typified by accumulations of heterochromatin protein 1 (HP1), methylation of histone H3 at lysine 9 (MeH3K9) and global histone deacetylation. HP1 interacts with chromatin by binding to MeH3K9 through the chromodomain (CD). HP1 dimerizes with itself and binds a variety of proteins through its chromoshadow domain. We have analyzed at the single cell level whether HP1 lacking its functional CD is able to induce heterochromatinization in vivo. We used a lac-operator array-based system in mammalian cells to target EGFP-lac repressor tagged truncated HP1alpha and HP1beta to a lac operator containing gene-amplified chromosome region in living cells. After targeting truncated HP1alpha or HP1beta we observe enhanced tri-MeH3K9 and recruitment of endogenous HP1alpha and HP1beta to the chromosome region. We show that CD-less HP1alpha can induce chromatin condensation, whereas the effect of truncated HP1beta is less pronounced. Our results demonstrate that after lac repressor-mediated targeting, HP1alpha and HP1beta without a functional CD are able to induce heterochromatinization. PMID:16283356

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

    PubMed Central

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

    2014-01-01

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

  10. 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

  11. 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

  12. In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

    PubMed Central

    Psaila, Bethan; Bussel, James B.; Linden, Matthew D.; Babula, Bracken; Li, Youfu; Barnard, Marc R.; Tate, Chinara; Mathur, Kanika; Frelinger, Andrew L.

    2012-01-01

    The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased. PMID:22294727

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

    PubMed

    Weber, Michael; Huisken, Jan

    2015-01-01

    Detailed studies of heart development and function are crucial for our understanding of cardiac failures and pave the way for better diagnostics and treatment. However, the constant motion and close incorporation into the cardiovascular system prevent in vivo studies of the living, unperturbed heart. The complementary strengths of the zebrafish model and light sheet microscopy provide a useful platform to fill this gap. High-resolution images of the embryonic vertebrate heart are now recorded from within the living animal: deep inside the unperturbed heart we can follow cardiac contractions and measure action potentials and calcium transients. Three-dimensional reconstructions of the entire beating heart with cellular resolution give new insights into its ever-changing morphology and facilitate studies into how individual cells form the complex cardiac network. In addition, cardiac dynamics and robustness are now examined with targeted optical manipulation. Overall, the combination of zebrafish and light sheet microscopy represents a promising addition for cardiac research and opens the door to a better understanding of heart function and development. PMID:26700795

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

    PubMed

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

    2012-03-01

    The primacy and power of RNA in governing many processes of life has begun to be more fully appreciated in both the discovery and inventive sciences. A variety of RNA interactions regulate gene expression, and structural self-assembly underlies many of these processes. The understanding sparked by these discoveries has inspired and informed the engineering of novel RNA structures, control elements, and genetic circuits in cells. Many of these engineered systems are built up fundamentally from RNA-RNA interactions, often combining modular, rational design with functional selection and screening. It is therefore useful to review the particular class of RNA-based regulatory mechanisms that rely on RNA self-assembly either through homomeric (self-self) or heteromeric (self-nonself) RNA-RNA interactions. Structures and sequence elements within individual RNAs create a basis for the pairing interactions, and in some instances can even lead to the formation of RNA polymers. Example systems of dimers, multimers, and polymers are reviewed in this article in the context of natural systems, wherein the function and impact of self-assemblies are understood. Following this, a brief overview is presented of specific engineered RNA self-assembly systems implemented in vivo, with lessons learned from both discovery and engineering approaches to RNA-RNA self-assembly. PMID:23914307

  15. 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.

  16. 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

  17. Predicting In Vivo Responses to Biomaterials via Combined In Vitro and In Silico Analysis

    PubMed Central

    Wolf, Matthew T.; Vodovotz, Yoram; Tottey, Stephen; Brown, Bryan N.

    2015-01-01

    The host response to both synthetic and biologically derived biomaterials is a temporally regulated, complex process that involves multiple interacting cell types. This complexity has classically limited the efficacy of in vitro assays for predicting the in vivo outcome, necessitating the use of costly animal models for biomaterial development. The present study addressed these challenges by developing an in vitro assay that characterized the dynamic inflammatory response of human monocyte-derived-macrophages to biomaterials, coupled with quasi-mechanistic analysis in silico analysis: principal component analysis (PCA) and dynamic network analysis (DyNA). Synthetic and extracellular matrix (ECM)–derived materials were evaluated using this method, and were then associated with the in vivo remodeling and macrophage polarization response in a rodent skeletal muscle injury model. PCA and DyNA revealed a distinct in vitro macrophage response to ECM materials that corresponded to constructive remodeling and an increased M2 macrophage presence in vivo. In contrast, PCA and DyNA suggested a response to crosslinked ECM and synthetic materials characteristic of a foreign body reaction and dominant M1 macrophage response. These results suggest that in silico analysis of an in vitro macrophage assay may be useful as a predictor for determining the in vivo host response to implanted biomaterials. PMID:24980950

  18. Separate and combined effects of a 10-d exposure to hypoxia and inactivity on oxidative function in vivo and mitochondrial respiration ex vivo in humans.

    PubMed

    Salvadego, Desy; Keramidas, Michail E; Brocca, Lorenza; Domenis, Rossana; Mavelli, Irene; Rittweger, Jörn; Eiken, Ola; Mekjavic, Igor B; Grassi, Bruno

    2016-07-01

    An integrative evaluation of oxidative metabolism was carried out in 9 healthy young men (age, 24.1 ± 1.7 yr mean ± SD) before (CTRL) and after a 10-day horizontal bed rest carried out in normoxia (N-BR) or hypoxia (H-BR, FiO2 = 0.147). H-BR was designed to simulate planetary habitats. Pulmonary O2 uptake (V̇o2) and vastus lateralis fractional O2 extraction (changes in deoxygenated hemoglobin+myoglobin concentration, Δ[deoxy(Hb+Mb)] evaluated using near-infrared spectroscopy) were evaluated in normoxia and during an incremental cycle ergometer (CE) and one-leg knee extension (KE) exercise (aimed at reducing cardiovascular constraints to oxidative function). Mitochondrial respiration was evaluated ex vivo by high-resolution respirometry in permeabilized vastus lateralis fibers. During CE V̇o2peak and Δ[deoxy(Hb+Mb)]peak were lower (P < 0.05) after both N-BR and H-BR than during CTRL; during KE the variables were lower after N-BR but not after H-BR. During CE the overshoot of Δ[deoxy(Hb+Mb)] during constant work rate exercise was greater in N-BR and H-BR than CTRL, whereas during KE a significant difference vs. CTRL was observed only after N-BR. Maximal mitochondrial respiration determined ex vivo was not affected by either intervention. In N-BR, a significant impairment of oxidative metabolism occurred downstream of central cardiovascular O2 delivery and upstream of mitochondrial function, possibly at the level of the intramuscular matching between O2 supply and utilization and peripheral O2 diffusion. Superposition of hypoxia on bed rest did not aggravate, and partially reversed, the impairment of muscle oxidative function in vivo induced by bed rest. The effects of longer exposures will have to be determined. PMID:27197861

  19. 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.

  20. 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

  1. A proposed multidimensional analysis function

    NASA Astrophysics Data System (ADS)

    Knight, Byron F.; Hamilton, Mark K.

    2003-08-01

    Previous work has suggested a potential value in the combination of physical property data types (e.g. magnetic and terrain slope) when searching for oil and mineral deposits. This work studies a notional multi-dimensional function to determine the likelihood of finding such deposits. Additionally, this hypothesis assumes some basic requirements must be meet in order to validate this function. The standard for determining the value of commercially gathered electro optical imagery is the same as with any optical system -- the ability to determine object in the field of view. Further, this function is defined as the ability to determine the presence of two parallel lines, vice only one. The National Imagery and Mapping Agency (NIMA) uses a function called Digital Terrain Elevation Data (DTED) to determine the elevation within a field of view. The DTED values for each pixel within a digital, commercial image can be considered similar to a gradient, whereby higher values are merely higher elevations. For the commercial electro optical system IKONOS (owned by Space Imaging, Inc.), the "resolution" is commonly referred to as 1 meter, which is the least discernable, parallel-line, separation distance. This hypothesis uses gravity and magnetic data to augment the DTED "gradient". As with the terrain values on the earth, gravity and magnetic values are continuously changing. Further, they can change for various reasons. Both are greatly affected by the changes in the subsurface materials, or the density of the soil and metallic content (e.g. iron). It is precisely these variations, through the combination of such differing forms of data, which can help determine the presence of oil and mineral deposits. The core of this work is a notional function development. Previous peer review has rightly pointed out that data fusion principles state that data must be commensurable before it can be fused. This work does not attempt to redefine data fusion concepts, but merely establish

  2. 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

  3. Transdifferentiation of Fast Skeletal Muscle Into Functional Endothelium in Vivo by Transcription Factor Etv2

    PubMed Central

    Gomez, Gustavo A.; Lindgren, Anne G.; Huang, Haigen; Yang, Hanshuo; Yao, Shaohua; Martin, Benjamin L.; Kimelman, David; Lin, Shuo

    2013-01-01

    Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly. PMID:23853546

  4. 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

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

    PubMed

    Schukur, Lina; Geering, Barbara; Fussenegger, Martin

    2016-03-01

    Encapsulated designer cells implanted into mice are currently used to validate the efficacy of therapeutic gene networks for the diagnosis and treatment of various human diseases in preclinical research. Because many human conditions cannot be adequately replicated by animal models, complementary and alternative procedures to test future treatment strategies are required. Here we describe a novel approach utilizing an ex vivo human whole-blood culture system to validate synthetic biology-inspired designer cell-based treatment strategies. The viability and functionality of transgenic mammalian designer cells co-cultured with primary human immune cells were characterized. We demonstrated that transgenic mammalian designer cells required adequate insulation from the human blood microenvironment to maintain viability and functionality. The biomaterial alginate-(poly-l-lysine)-alginate used to encapsulate the transgenic designer cells did neither affect the viability of primary granulocytes and lymphocytes nor the functionality of lymphocytes. Additionally, alginate-encapsulated transgenic designer cells remained responsive to the release of the pro-inflammatory cytokine tumor necrosis factor (TNF) from the whole-blood culture upon exposure to bacterial lipopolysaccharide (LPS). TNF diffused into the alginate capsules, bound to the specific TNF receptors on the transgenic designer cells' surface and triggered the expression of the reporter gene SEAP (human placental secreted alkaline phosphatase) that was rewired to the TNF-specific signaling cascade. Human whole-blood culture systems can therefore be considered as valuable complementary assays to animal models for the validation of synthetic circuits in genetically modified mammalian cells and may speed up preclinical research in a world of personalized medicine. PMID:26348251

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

    PubMed

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

    2011-01-01

    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

  7. 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

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

    PubMed

    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 6 h. After 24 h kidney function was measured via Inulin- and PAH-Clearance in re-anesthetized rats, and serum urea, creatinine (crea), cystatin C and Neutrophil gelatinase-associated lipocalin (NGAL) as well as histopathology were analysed. In vitro human proximal tubule cells (PTC) were cultured +/- lipopolysaccharid (LPS) and with 0.1-4.0% VOL. Cell viability was measured with XTT-, cell toxicity with LDH-test. sCASP induced severe septic AKI demonstrated divergent results regarding renal function by clearance or creatinine measure focusing on VOL. Soleley HES (C+VOL) deteriorated renal function without sCASP. Histopathology revealed significantly derangements in all HES groups compared to control. In vitro LPS did not worsen the HES induced reduction of cell viability in PTC cells. For the first time, we demonstrated, that application of 50 ml/KG 6% HES 130/0.4 over 6 hours induced AKI without inflammation in vivo. Severity of sCASP induced septic AKI might be no longer susceptible to the way of volume expansion. PMID:26340751

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

    PubMed

    Brenneis, Mariam; Soppa, Jörg

    2009-01-01

    Recently a first genome-wide analysis of translational regulation using prokaryotic species had been performed which revealed that regulation of translational efficiency plays an important role in haloarchaea. In fact, the fractions of genes under differential growth phase-dependent translational control in the two species Halobacterium salinarum and Haloferax volcanii were as high as in eukaryotes. However, nothing is known about the mechanisms of translational regulation in archaea. Therefore, two genes exhibiting opposing directions of regulation were selected to unravel the importance of untranslated regions (UTRs) for differential translational control in vivo.Differential translational regulation in exponentially growing versus stationary phase cells was studied by comparing translational efficiencies using a reporter gene system. Translational regulation was not observed when 5'-UTRs or 3'-UTRs alone were fused to the reporter gene. However, their simultaneous presence was sufficient to transfer differential translational control from the native transcript to the reporter transcript. This was true for both directions of translational control. Translational regulation was completely abolished when stem loops in the 5'-UTR were changed by mutagenesis. An "UTR-swap" experiment demonstrated that the direction of translational regulation is encoded in the 3'-UTR, not in the 5'-UTR. While much is known about 5'-UTR-dependent translational control in bacteria, the reported findings provide the first examples that both 5'- and 3'-UTRs are essential and sufficient to drive differential translational regulation in a prokaryote and therefore have to functionally interact in vivo. The current results indicate that 3'-UTR-dependent translational control had already evolved before capping and polyadenylation of transcripts were invented, which are essential for circularization of transcripts in eukaryotes. PMID:19214227

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

    NASA Astrophysics Data System (ADS)

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

    2011-10-01

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