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Sample records for in-vivo lacr-mediated gene

  1. Visually Relating Gene Expression and in vivo DNA Binding Data

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Min-Yu; Mackey, Lester; Ker?,; nen, Soile V. E.; Weber, Gunther H.; Jordan, Michael I.; Knowles, David W.; Biggin, Mark D.; Hamann, Bernd

    2011-09-20

    Gene expression and in vivo DNA binding data provide important information for understanding gene regulatory networks: in vivo DNA binding data indicate genomic regions where transcription factors are bound, and expression data show the output resulting from this binding. Thus, there must be functional relationships between these two types of data. While visualization and data analysis tools exist for each data type alone, there is a lack of tools that can easily explore the relationship between them. We propose an approach that uses the average expression driven by multiple of ciscontrol regions to visually relate gene expression and in vivo DNA binding data. We demonstrate the utility of this tool with examples from the network controlling early Drosophila development. The results obtained support the idea that the level of occupancy of a transcription factor on DNA strongly determines the degree to which the factor regulates a target gene, and in some cases also controls whether the regulation is positive or negative.

  2. In Vivo Imaging of mdrla Gene Expression

    National Research Council Canada - National Science Library

    Synold, Timothy W

    2005-01-01

    .... With the advent of new bioimaging technology and the advancement of efficient gene targeting strategies, they found an opportunity to apply these state-of-the-art molecular tools to their problem...

  3. Highly Effective Gene Transfection In Vivo by Alkylated Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Jennifer A. Fortune

    2011-01-01

    Full Text Available We mechanistically explored the effect of increased hydrophobicity of the polycation on the efficacy and specificity of gene delivery in mice. N-Alkylated linear PEIs with varying alkyl chain lengths and extent of substitution were synthesized and characterized by biophysical methods. Their in vivo transfection efficiency, specificity, and biodistribution were investigated. N-Ethylation improves the in vivo efficacy of gene expression in the mouse lung 26-fold relative to the parent polycation and more than quadruples the ratio of expression in the lung to that in all other organs. N-Propyl-PEI was the best performer in the liver and heart (581- and 3.5-fold enhancements, resp. while N-octyl-PEI improved expression in the kidneys over the parent polymer 221-fold. As these enhancements in gene expression occur without changing the plasmid biodistribution, alkylation does not alter the cellular uptake but rather enhances transfection subsequent to cellular uptake.

  4. Sustainability of keratinocyte gene transfer and cell survival in vivo.

    Science.gov (United States)

    Choate, K A; Khavari, P A

    1997-05-20

    The epidermis is an attractive site for therapeutic gene delivery because it is accessible and capable of delivering polypeptides to the systemic circulation. A number of difficulties, however, have emerged in attempts at cutaneous gene delivery, and central among these is an inability to sustain therapeutic gene production. We have examined two major potential contributing factors, viral vector stamina and involvement of long-lived epidermal progenitor cells. Human keratinocytes were either untreated or transduced with a retroviral vector for beta-galactosidase (beta-Gal) at > 99% efficiency and then grafted onto immunodeficient mice to regenerate human epidermis. Human epidermis was monitored in vivo after grafting for clinical and histologic appearance as well as for gene expression. Although integrated vector sequences persisted unchanged in engineered epidermis at 10 weeks post-grafting, retroviral long terminal repeat (LTR)-driven beta-Gal expression ceased in vivo after approximately 4 weeks. Endogenous cellular promoters, however, maintained consistently normal gene expression levels without evidence of time-dependent decline, as determined by immunostaining with species-specific antibodies for human involucrin, filaggrin, keratinocyte transglutaminase, keratin 10, type VII collagen, and Laminin 5 proteins out to week 14 post-grafting. Transduced human keratinocytes generated multilayer epidermis sustained through multiple epidermal turnover cycles; this epidermis demonstrated retention of a spatially appropriate pattern of basal and suprabasal epidermal marker gene expression. These results confirm previous findings suggesting that viral promoter-driven gene expression is not durable and demonstrate that keratinocytes passaged in vitro can regenerate and sustain normal epidermis for prolonged periods.

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

    Science.gov (United States)

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

    1999-07-01

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

  6. Theory and in vivo application of electroporative gene delivery.

    Science.gov (United States)

    Somiari, S; Glasspool-Malone, J; Drabick, J J; Gilbert, R A; Heller, R; Jaroszeski, M J; Malone, R W

    2000-09-01

    Efficient and safe methods for delivering exogenous genetic material into tissues must be developed before the clinical potential of gene therapy will be realized. Recently, in vivo electroporation has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines (NAV). Electroporation (EP) involves the application of pulsed electric fields to cells to enhance cell permeability, resulting in exogenous polynucleotide transit across the cytoplasmic membrane. Similar pulsed electrical field treatments are employed in a wide range of biotechnological processes including in vitro EP, hybridoma production, development of transgenic animals, and clinical electrochemotherapy. Electroporative gene delivery studies benefit from well-developed literature that may be used to guide experimental design and interpretation. Both theory and experimental analysis predict that the critical parameters governing EP efficacy include cell size and field strength, duration, frequency, and total number of applied pulses. These parameters must be optimized for each tissue in order to maximize gene delivery while minimizing irreversible cell damage. By providing an overview of the theory and practice of electroporative gene transfer, this review intends to aid researchers that wish to employ the method for preclinical and translational gene therapy, NAV, and functional genomic research.

  7. In vivo gene expression and immunoreactivity of Leptospira collagenase.

    Science.gov (United States)

    Janwitthayanan, Weena; Keelawat, Somboon; Payungporn, Sunchai; Lowanitchapat, Alisa; Suwancharoen, Duangjai; Poovorawan, Yong; Chirathaworn, Chintana

    2013-06-12

    Pulmonary hemorrhage is an increasing cause of death of leptospirosis patients. Bacterial collagenase has been shown to be involved in lung hemorrhage induced by various infectious agents. According to Leptospira whole genome study, colA, a gene suggested to code for bacterial collagenase has been identified. We investigated colA gene expression in lung tissues of Leptospira infected hamsters. Golden Syrian Hamsters were injected intraperitoneally with Leptospira interrogans serovar Pyrogenes. The hamsters were sacrificed on days 3, 5 and 7 post-infection and lung tissues were collected for histological examination and RNA extraction. Lung pathologies including atelectasis and hemorrhage were observed. Expression of colA gene in lung tissues was demonstrated by both RT-PCR and real time PCR. In addition, ColA protein was cloned and the purified protein could react with sera from leptospirosis patients. Leptospira ColA protein may play a role in Leptospira survival or pathogenesis in vivo. Its reaction with leptospirosis sera suggests that this protein is immunogenic and could be another candidate for vaccine development. Copyright © 2012 Elsevier GmbH. All rights reserved.

  8. GENE TRANSFER IN TOBACCO MITOCHONDRIA IN VITRO AND IN VIVO

    Directory of Open Access Journals (Sweden)

    Katyshev A.I.

    2012-08-01

    Full Text Available Earlier, we had showed that isolated mitochondria from different organisms can import DNA. Exploiting this mechanism, we assessed the possibility of genes transfer in tobacco mitochondria in vitro and in vivo. Whereas homologous recombination is a rare occasion in higher plant nuclei, recombination between the large direct repeats in plant mitochondrial genome generates its multipartite structure. Following transfection of isolated organelles with constructs composed of a partial gfp gene flanked by mitochondrial DNA fragments, we showed the homologous recombination of imported DNA with the resident DNA and the integration of the reporter gene. The recombination yielded an insertion of a continuous exogenous DNA fragment including the gfp sequence and at least the 0.5 kb of the flanking sequence on each side. Using of transfection constructs carrying multiple sequences homologous to mitochondrial DNA could be suitable for insertion of a target gene into any region of the mitochondrial genome, which turns this approach to be of a general and methodical importance. Usually mitochondrial reactive oxygen species (ROS level is under strict control of the antioxidant system including the Mn-containing superoxide dismutase (MnSOD. MnSOD is presented in multiple forms encoded by several genes in plants. Possibly, this enzyme, beside its catalytic function, fulfills as well some unknown biochemical functions. Thus, one of maize SOD enzymes (SOD3.4 could bind with mitochondrial DNA. Another SOD form (SOD3.1 is located in close proximity to mitochondrial respiratory complexes, where ROS are generated. To study possible physiological functions of this enzyme, we cloned the maize SOD3.1 gene. Compared to the SOD3.4, this enzyme didn't demonstrate DNA-binding activity. At the same time, SOD3.1 didn't show non-specific DNA-hydrolyzing activity as Cu/ZnSOD does. It means that this enzyme might have some DNA protective function. We made NtPcob-sod3.1-IGR

  9. Manipulating the in vivo immune response by targeted gene knockdown.

    Science.gov (United States)

    Lieberman, Judy

    2015-08-01

    Aptamers, nucleic acids selected for high affinity binding to proteins, can be used to activate or antagonize immune mediators or receptors in a location and cell-type specific manner and to enhance antigen presentation. They can also be linked to other molecules (other aptamers, siRNAs or miRNAs, proteins, toxins) to produce multifunctional compounds for targeted immune modulation in vivo. Aptamer-siRNA chimeras (AsiCs) that induce efficient cell-specific knockdown in immune cells in vitro and in vivo can be used as an immunological research tool or potentially as an immunomodulating therapeutic. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. In vivo SPECT reporter gene imaging of regulatory T cells.

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    Ehsan Sharif-Paghaleh

    Full Text Available Regulatory T cells (Tregs were identified several years ago and are key in controlling autoimmune diseases and limiting immune responses to foreign antigens, including alloantigens. In vivo imaging techniques including intravital microscopy as well as whole body imaging using bioluminescence probes have contributed to the understanding of in vivo Treg function, their mechanisms of action and target cells. Imaging of the human sodium/iodide symporter via Single Photon Emission Computed Tomography (SPECT has been used to image various cell types in vivo. It has several advantages over the aforementioned imaging techniques including high sensitivity, it allows non-invasive whole body studies of viable cell migration and localisation of cells over time and lastly it may offer the possibility to be translated to the clinic. This study addresses whether SPECT/CT imaging can be used to visualise the migratory pattern of Tregs in vivo. Treg lines derived from CD4(+CD25(+FoxP3(+ cells were retrovirally transduced with a construct encoding for the human Sodium Iodide Symporter (NIS and the fluorescent protein mCherry and stimulated with autologous DCs. NIS expressing self-specific Tregs were specifically radiolabelled in vitro with Technetium-99m pertechnetate ((99mTcO(4(- and exposure of these cells to radioactivity did not affect cell viability, phenotype or function. In addition adoptively transferred Treg-NIS cells were imaged in vivo in C57BL/6 (BL/6 mice by SPECT/CT using (99mTcO(4(-. After 24 hours NIS expressing Tregs were observed in the spleen and their localisation was further confirmed by organ biodistribution studies and flow cytometry analysis. The data presented here suggests that SPECT/CT imaging can be utilised in preclinical imaging studies of adoptively transferred Tregs without affecting Treg function and viability thereby allowing longitudinal studies within disease models.

  11. Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques

    Directory of Open Access Journals (Sweden)

    Daniell Henry

    2011-06-01

    Full Text Available Abstract Background Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun" delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI methods. Results Plasmid DNA carrying the firefly luciferase (LUC reporter gene under the control of the human Cytomegalovirus (CMV promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter using different DNA Loading Ratios (DLRs, and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50 at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. Conclusions The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results

  12. In vivo expression of genes in the entomopathogenic fungus Beauveria bassiana during infection of lepidopteran larvae.

    Science.gov (United States)

    Galidevara, Sandhya; Reineke, Annette; Koduru, Uma Devi

    2016-05-01

    The entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin is commercially available as a bio insecticide. The expression of three genes previously identified to have a role in pathogenicity in in vitro studies was validated in vivo in three lepidopteran insects infected with B. bassiana. Expression of all three genes was observed in all the tested insects starting from 48 or 72h to 10d post infection corroborating their role in pathogenicity. We suggest that it is essential to test the expression of putative pathogenicity genes both in vitro and in vivo to understand their role in different insect species. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. In Vivo Gene Therapy of Hemophilia B: Sustained Partial Correction in Factor IX-Deficient Dogs

    Science.gov (United States)

    Kay, Mark A.; Rothenberg, Steven; Landen, Charles N.; Bellinger, Dwight A.; Leland, Frances; Toman, Carol; Finegold, Milton; Thompson, Arthur R.; Read, M. S.; Brinkhous, Kenneth M.; Woo, Savio L. C.

    1993-10-01

    The liver represents a model organ for gene therapy. A method has been developed for hepatic gene transfer in vivo by the direct infusion of recombinant retroviral vectors into the portal vasculature, which results in the persistent expression of exogenous genes. To determine if these technologies are applicable for the treatment of hemophilia B patients, preclinical efficacy studies were done in a hemophilia B dog model. When the canine factor IX complementary DNA was transduced directly into the hepatocytes of affected dogs in vivo, the animals constitutively expressed low levels of canine factor IX for more than 5 months. Persistent expression of the clotting. factor resulted in reductions of whole blood clotting and partial thromboplastin times of the treated animals. Thus, long-term treatment of hemophilia B patients may be feasible by direct hepatic gene therapy in vivo.

  14. Garlic Influences Gene Expression In Vivo and In Vitro.

    Science.gov (United States)

    Charron, Craig S; Dawson, Harry D; Novotny, Janet A

    2016-02-01

    There is a large body of preclinical research aimed at understanding the roles of garlic and garlic-derived preparations in the promotion of human health. Most of this research has targeted the possible functions of garlic in maintaining cardiovascular health and in preventing and treating cancer. A wide range of outcome variables has been used to investigate the bioactivity of garlic, ranging from direct measures of health status such as cholesterol concentrations, blood pressure, and changes in tumor size and number, to molecular and biochemical measures such as mRNA gene expression, protein concentration, enzyme activity, and histone acetylation status. Determination of how garlic influences mRNA gene expression has proven to be a valuable approach to elucidating the mechanisms of garlic bioactivity. Preclinical studies investigating the health benefits of garlic far outnumber human studies and have made frequent use of mRNA gene expression measurement. There is an immediate need to understand mRNA gene expression in humans as well. Although safety and ethical constraints limit the types of available human tissue, peripheral whole blood is readily accessible, and measuring mRNA gene expression in whole blood may provide a unique window to understanding how garlic intake affects human health. © 2016 American Society for Nutrition.

  15. Polymeric Nanoparticles for Nonviral Gene Therapy Extend Brain Tumor Survival in Vivo

    OpenAIRE

    Mangraviti, Antonella; Tzeng, Stephany Yi; Kozielski, Kristen Lynn; Wang, Yuan; Jin, Yike; Gullotti, David; Pedone, Mariangela; Buaron, Nitsa; Liu, Ann; Wilson, David R.; Hansen, Sarah K.; Rodriguez, Fausto J.; Gao, Guo-Dong; DiMeco, Francesco; Brem, Henry

    2015-01-01

    Biodegradable polymeric nanoparticles have the potential to be safer alternatives to viruses for gene delivery; however, their use has been limited by poor efficacy in vivo. In this work, we synthesize and characterize polymeric gene delivery nanoparticles and evaluate their efficacy for DNA delivery of herpes simplex virus type I thymidine kinase (HSVtk) combined with the prodrug ganciclovir (GCV) in a malignant glioma model. We investigated polymer structure for gene delivery in two rat gli...

  16. Differential in vivo gene expression of major Leptospira proteins in resistant or susceptible animal models.

    Science.gov (United States)

    Matsui, Mariko; Soupé, Marie-Estelle; Becam, Jérôme; Goarant, Cyrille

    2012-09-01

    Transcripts of Leptospira 16S rRNA, FlaB, LigB, LipL21, LipL32, LipL36, LipL41, and OmpL37 were quantified in the blood of susceptible (hamsters) and resistant (mice) animal models of leptospirosis. We first validated adequate reference genes and then evaluated expression patterns in vivo compared to in vitro cultures. LipL32 expression was downregulated in vivo and differentially regulated in resistant and susceptible animals. FlaB expression was also repressed in mice but not in hamsters. In contrast, LigB and OmpL37 were upregulated in vivo. Thus, we demonstrated that a virulent strain of Leptospira differentially adapts its gene expression in the blood of infected animals.

  17. Identification of Spt5 target genes in zebrafish development reveals its dual activity in vivo.

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    Keerthi Krishnan

    Full Text Available Spt5 is a conserved essential protein that represses or stimulates transcription elongation in vitro. Immunolocalization studies on Drosophila polytene chromosomes suggest that Spt5 is associated with many loci throughout the genome. However, little is known about the prevalence and identity of Spt5 target genes in vivo during development. Here, we identify direct target genes of Spt5 using fog(sk8 zebrafish mutant, which disrupts the foggy/spt5 gene. We identified that fog(sk8 and their wildtype siblings differentially express less than 5% of genes examined. These genes participate in diverse biological processes from stress response to cell fate specification. Up-regulated genes exhibit shorter overall gene length compared to all genes examined. Through chromatin immunoprecipitation in zebrafish embryos, we identified a subset of developmentally critical genes that are bound by both Spt5 and RNA polymerase II. The protein occupancy patterns on these genes are characteristic of both repressive and stimulatory elongation regulation. Together our findings establish Spt5 as a dual regulator of transcription elongation in vivo and identify a small but diverse set of target genes critically dependent on Spt5 during development.

  18. Distinct C/EBPalpha motifs regulate lipogenic and gluconeogenic gene expression in vivo

    DEFF Research Database (Denmark)

    Pedersen, Thomas A; Bereshchenko, Oxana; Garcia-Silva, Susana

    2007-01-01

    gluconeogenesis and lipogenesis. In vivo deletion of a proline-histidine rich domain (PHR), dephosphorylated at S193 by insulin signaling, dysregulated genes involved in the generation of acetyl-CoA and NADPH for triglyceride synthesis and led to increased hepatic lipogenesis. These promoters bound SREBP-1...

  19. Transcription factor 7-like 2 gene links increased in vivo insulin synthesis to type 2 diabetes

    NARCIS (Netherlands)

    S. Jainandunsing (Sjaam); Koole, H.R. (H. Rita); van Miert, J.N.I. (Joram N.I.); T. Rietveld (Trinet); J.L.D. Wattimena (Josias); E.J.G. Sijbrands (Eric); F.W.M. de Rooij (Felix)

    2018-01-01

    textabstractTranscription factor 7-like 2 (TCF7L2) is the main susceptibility gene for type 2 diabetes, primarily through impairing the insulin secretion by pancreatic β cells. However, the exact in vivo mechanisms remain poorly understood. We performed a family study and determined if the T risk

  20. In Vivo Imaging of Local Gene Expression Induced by Magnetic Hyperthermia

    Directory of Open Access Journals (Sweden)

    Olivier Sandre

    2017-02-01

    Full Text Available The present work aims to demonstrate that colloidal dispersions of magnetic iron oxide nanoparticles stabilized with dextran macromolecules placed in an alternating magnetic field can not only produce heat, but also that these particles could be used in vivo for local and noninvasive deposition of a thermal dose sufficient to trigger thermo-induced gene expression. Iron oxide nanoparticles were first characterized in vitro on a bio-inspired setup, and then they were assayed in vivo using a transgenic mouse strain expressing the luciferase reporter gene under transcriptional control of a thermosensitive promoter. Iron oxide nanoparticles dispersions were applied topically on the mouse skin or injected subcutaneously with Matrigel™ to generate so-called pseudotumors. Temperature was monitored continuously with a feedback loop to control the power of the magnetic field generator and to avoid overheating. Thermo-induced luciferase expression was followed by bioluminescence imaging 6 h after heating. We showed that dextran-coated magnetic iron oxide nanoparticle dispersions were able to induce in vivo mild hyperthermia compatible with thermo-induced gene expression in surrounding tissues and without impairing cell viability. These data open new therapeutic perspectives for using mild magnetic hyperthermia as noninvasive modulation of tumor microenvironment by local thermo-induced gene expression or drug release.

  1. Cell surface expression of single chain antibodies with applications to imaging of gene expression in vivo

    International Nuclear Information System (INIS)

    Northrop, Jeffrey P.; Bednarski, Mark; Li, King C.; Barbieri, Susan O.; Lu, Amy T.; Nguyen, Dee; Varadarajan, John; Osen, Maureen; Star-Lack, Josh

    2003-01-01

    Imaging of gene expression in vivo has many potential uses for biomedical research and drug discovery, ranging from the study of gene regulation and cancer to the non-invasive assessment of gene therapies. To streamline the development of imaging marker gene technologies for nuclear medicine, we propose a new approach to the design of reporter/probe pairs wherein the reporter is a cell surface-expressed single chain antibody variable fragment that has been raised against a low molecular weight imaging probe with optimized pharmacokinetic properties. Proof of concept of the approach was achieved using a single chain antibody variable fragment that binds with high affinity to fluorescein and an imaging probe consisting of fluorescein isothiocyanate coupled to the chelator diethylene triamine penta-acetic acid labeled with the gamma-emitter 111 In. We demonstrate specific high-affinity binding of this probe to the cell surface-expressed reporter in vitro and assess the in vivo biodistribution of the probe both in wild-type mice and in mice harboring tumor xenografts expressing the reporter. Specific uptake of the probe by, and in vivo imaging of, tumors expressing the reporter are shown. Since ScFvs with high affinities can be raised to almost any protein or small molecule, the proposed methodology may offer a new flexibility in the design of imaging tracer/reporter pairs wherein both probe pharmacokinetics and binding affinities can be readily optimized. (orig.)

  2. Positron emission tomography and gene therapy: basic concepts and experimental approaches for in vivo gene expression imaging.

    Science.gov (United States)

    Peñuelas, Iván; Boán, JoséF; Martí-Climent, Josep M; Sangro, Bruno; Mazzolini, Guillermo; Prieto, Jesús; Richter, José A

    2004-01-01

    More than two decades of intense research have allowed gene therapy to move from the laboratory to the clinical setting, where its use for the treatment of human pathologies has been considerably increased in the last years. However, many crucial questions remain to be solved in this challenging field. In vivo imaging with positron emission tomography (PET) by combination of the appropriate PET reporter gene and PET reporter probe could provide invaluable qualitative and quantitative information to answer multiple unsolved questions about gene therapy. PET imaging could be used to define parameters not available by other techniques that are of substantial interest not only for the proper understanding of the gene therapy process, but also for its future development and clinical application in humans. This review focuses on the molecular biology basis of gene therapy and molecular imaging, describing the fundamentals of in vivo gene expression imaging by PET, and the application of PET to gene therapy, as a technology that can be used in many different ways. It could be applied to avoid invasive procedures for gene therapy monitoring; accurately diagnose the pathology for better planning of the most adequate therapeutic approach; as treatment evaluation to image the functional effects of gene therapy at the biochemical level; as a quantitative noninvasive way to monitor the location, magnitude and persistence of gene expression over time; and would also help to a better understanding of vector biology and pharmacology devoted to the development of safer and more efficient vectors.

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

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    Minyan Li

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

  4. Gene delivery to pancreatic exocrine cells in vivo and in vitro

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    Houbracken Isabelle

    2012-10-01

    Full Text Available Abstract Background Effective gene transfer to the pancreas or to pancreatic cells has remained elusive although it is essential for studies of genetic lineage tracing and modulation of gene expression. Different transduction methods and viral vectors were tested in vitro and in vivo, in rat and mouse pancreas. Results For in vitro transfection/transduction of rat exocrine cells lipofection reagents, adenoviral vectors, and Mokola- and VSV-G pseudotyped lentiviral vectors were used. For in vivo transduction of mouse and rat pancreas adenoviral vectors and VSV-G lentiviral vectors were injected into the parenchymal tissue. Both lipofection of rat exocrine cell cultures and transduction with Mokola pseudotyped lentiviral vectors were inefficient and resulted in less than 4% EGFP expressing cells. Adenoviral transduction was highly efficient but its usefulness for gene delivery to rat exocrine cells in vitro was hampered by a drastic increase in cell death. In vitro transduction of rat exocrine cells was most optimal with VSV-G pseudotyped lentiviral vectors, with stable transgene expression, no significant effect on cell survival and about 40% transduced cells. In vivo, pancreatic cells could not be transduced by intra-parenchymal administration of lentiviral vectors in mouse and rat pancreas. However, a high efficiency could be obtained by adenoviral vectors, resulting in transient transduction of mainly exocrine acinar cells. Injection in immune-deficient animals diminished leukocyte infiltration and prolonged transgene expression. Conclusions In summary, our study remarkably demonstrates that transduction of pancreatic exocrine cells requires lentiviral vectors in vitro but adenoviral vectors in vivo.

  5. In Vitro and In Vivo Effective Gene Delivery with Novel Liposomal Bubbles

    Science.gov (United States)

    Nishiie, Norihito; Suzuki, Ryo; Oda, Yusuke; Hirata, Keiichi; Taira, Yuichiro; Utoguchi, Naoki; Negishi, Yoichi; Maruyama, Kazuo

    2010-03-01

    Microbubbles, which were ultrasound contrast agents, could improve the transfection efficiency by cavitation with ultrasound exposure. However, conventional microbubbles had some problems regarding size and targeting ability. To solve these problems, we paid attention to liposomes that had many advantages as drug, antigen and gene delivery carriers. Because they can easily be controlled their size and added a targeting function. And we succeeded to prepare novel liposomal bubbles (Bubble liposomes) entrapping perfluoropropane which was utilized for contrast enhancement in ultrasonography. In this study, we assessed the feasibility of Bubble liposomes as gene delivery tools utilized cavitation by ultrasound exposure. In vitro gene delivery, Bubble liposomes could deliver plasmid DNA to many cell types such as tumor cells, T cell line and endothelial cells without cytotoxicity. In vivo gene delivery, Bubble liposomes could effectively deliver plasmid DNA into mouse femoral artery. This method was more effectively than conventional lipofection. We conclude that Bubble liposomes are unique and efficient gene delivery tools in vitro and in vivo.

  6. Improved in vivo gene transfer into tumor tissue by stabilization of pseudodendritic oligoethylenimine-based polyplexes.

    Science.gov (United States)

    Russ, Verena; Fröhlich, Thomas; Li, Yunqiu; Halama, Anna; Ogris, Manfred; Wagner, Ernst

    2010-02-01

    HD O is a low molecular weight pseudodendrimer containing oligoethylenimine and degradable hexanediol diacrylate diesters. DNA polyplexes display encouraging gene transfer efficiency in vitro and in vivo but also a limited stability under physiological conditions. This limitation must be overcome for further development into more sophisticated formulations. HD O polyplexes were laterally stabilized by crosslinking surface amines via bifunctional crosslinkers, bioreducible dithiobis(succimidyl propionate) (DSP) or the nonreducible analog disuccinimidyl suberate (DSS). Optionally, in a subsequent step, the targeting ligand transferrin (Tf) was attached to DSP-linked HD O polyplexes via Schiff base formation between HD O amino groups and Tf aldehyde groups, which were introduced into Tf by periodate oxidation of the glycosylation sites. Crosslinked DNA polyplexes showed an increased stability against exchange reaction by salt or heparin. Disulfide bond containing DSP-linked polyplexes were susceptible to reducing conditions. These polyplexes displayed the highest gene expression levels in vitro and in vivo (upon intratumoral application in mice), and these were significantly elevated and prolonged over standard or DSS-stabilized HD O formulations. DSP-stabilized HD O polyplexes with or without Tf coating were well-tolerated after intravenous application. High gene expression levels were found in tumor tissue, with negligible gene expression in any other organ. Lateral stabilization of HD O polyplexes with DSP crosslinker enhanced gene transfer efficacy and was essential for the incorporation of a ligand (Tf) into a stable particle formulation.

  7. Cellular Immune Response Against Firefly Luciferase After Sleeping Beauty–Mediated Gene Transfer In Vivo

    Science.gov (United States)

    Podetz-Pedersen, Kelly M.; Vezys, Vaiva; Somia, Nikunj V.; Russell, Stephen J.

    2014-01-01

    Abstract The Sleeping Beauty (SB) transposon system has been shown to mediate new gene sequence integration resulting in long-term expression. Here the effectiveness of hyperactive SB100X transposase was tested, and we found that hydrodynamic co-delivery of a firefly luciferase transposon (pT2/CaL) along with SB100X transposase (pCMV-SB100X) resulted in remarkably sustained, high levels of luciferase expression. However, after 4 weeks there was a rapid, animal-by-animal loss of luciferase expression that was not observed in immunodeficient mice. We hypothesized that this sustained, high-level luciferase expression achieved using the SB100X transposase elicits an immune response in pT2/CaL co-administered mice, which was supported by the rapid loss of luciferase expression upon challenge of previously treated animals and in naive animals adoptively transferred with splenocytes from previously treated animals. Specificity of the immune response to luciferase was demonstrated by increased cytokine expression in splenocytes after exposure to luciferase peptide in parallel with MHC I–luciferase peptide tetramer binding. This anti-luciferase immune response observed following continuous, high-level luciferase expression in vivo clearly impacts its use as an in vivo reporter. As both an immunogen and an extremely sensitive reporter, luciferase is also a useful model system for the study of immune responses following in vivo gene transfer and expression. PMID:25093708

  8. Non-viral gene therapy that targets motor neurons in vivo

    Directory of Open Access Journals (Sweden)

    Mary-Louise eRogers

    2014-10-01

    Full Text Available A major challenge in neurological gene therapy is safe delivery of transgenes to sufficient cell numbers from the circulation or periphery. This is particularly difficult for diseases involving spinal cord motor neurons such as amyotrophic lateral sclerosis (ALS. We have examined the feasibility of non-viral gene delivery to spinal motor neurons from intraperitoneal injections of plasmids carried by ‘immunogene’ nanoparticles targeted for axonal retrograde transport using antibodies. PEGylated polyethylenimine (PEI-PEG12 as DNA carrier was conjugated to an antibody (MLR2 to the neurotrophin receptor p75 (p75NTR. We used a plasmid (pVIVO2 designed for in vivo gene delivery that produces minimal immune responses, has improved nuclear entry into post mitotic cells and also expresses green fluorescent protein (GFP. MLR2-PEI-PEG12 carried pVIVO2 and was specific for mouse motor neurons in mixed cultures containing astrocytes. While only 8% of motor neurons expressed GFP 72 h post transfection in vitro, when the immunogene was given intraperitonealy to neonatal C57BL/6J mice GFP specific motor neuron expression was observed in 25.4% of lumbar, 18.3% of thoracic and 17.0 % of cervical motor neurons, 72 h post transfection. PEI-PEG12 carrying pVIVO2 by itself did not transfect motor neurons in vivo, demonstrating the need for specificity via the p75NTR antibody MLR2. This is the first time that specific transfection of spinal motor neurons has been achieved from peripheral delivery of plasmid DNA as part of a non-viral gene delivery agent. These results stress the specificity and feasibility of immunogene delivery targeted for p75NTR expressing motor neurons, but suggests that further improvements are required to increase the transfection efficiency of motor neurons in vivo.

  9. Feasibility of baculovirus-mediated reporter gene delivery for efficient monitoring of islet transplantation in vivo

    International Nuclear Information System (INIS)

    Liu, Shuai; Pan, Yu; Lv, Jing; Wu, Haifei; Tian, Jingyan; Zhang, Yifan

    2014-01-01

    Objective: The objective of this study was to explore the feasibility of baculovirus vector-mediated sodium iodide symporter (NIS) gene delivery to monitor islet transplantation. Methods: Baculovirus vectors expressing green fluorescent protein (GFP) or NIS (Bac-GFP and Bac-NIS) were established using the Bac-to-Bac baculovirus expression system. The GFP expression of Bac-GFP-infected rat islets was observed in vitro by fluorescence microscopy. Iodine uptake and inhibition of iodine uptake by NaClO 4 in Bac-NIS-infected islets were dynamically monitored in vitro. Bac-GFP- or Bac-NIS-infected islets were implanted into the left axillary cavity of NOD-SCID mice, and fluorescence imaging and 125 I NanoSPECT/CT imaging were subsequently performed in vivo. Results: Bac-GFP efficiently infected rat islets (over 95% infected at MOI = 40), and the expression of GFP lasted approximately two weeks. NaClO 4 could inhibit iodine uptake by Bac-NIS-infected islets. In vivo imaging revealed that the fluorescence intensity of the transplant sites in Bac-GFP-infected groups was significantly higher than in the non-infected group. Grafts could be clearly observed by 125 I NanoSPECT/CT imaging for up to 8 h. Conclusion: Baculovirus vectors are powerful vehicles for studying rat islets in gene delivery. It is feasible to use a baculovirus vector to delivery an NIS gene for non-invasive monitoring transplanted islets in vivo by the expression of the target gene

  10. Leu452His mutation in lipoprotein lipase gene transfer associated with hypertriglyceridemia in mice in vivo.

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    Kaiyue Sun

    Full Text Available Mutated mouse lipoprotein lipase (LPL containing a leucine (L to histidine (H substitution at position 452 was transferred into mouse liver by hydrodynamics-based gene delivery (HD. Mutated-LPL (MLPL gene transfer significantly increased the concentrations of plasma MLPL and triglyceride (TG but significantly decreased the activity of plasma LPL. Moreover, the gene transfer caused adiposis hepatica and significantly increased TG content in mouse liver. To understand the effects of MLPL gene transfer on energy metabolism, we investigated the expression of key functional genes related to energy metabolism in the liver, epididymal fat, and leg muscles. The mRNA contents of hormone-sensitive lipase (HSL, adipose triglyceride lipase (ATGL, fatty acid-binding protein (FABP, and uncoupling protein (UCP were found to be significantly reduced. Furthermore, we investigated the mechanism by which MLPL gene transfer affected fat deposition in the liver, fat tissue, and muscle. The gene expression and protein levels of forkhead Box O3 (FOXO3, AMP-activated protein kinase (AMPK, and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α were found to be remarkably decreased in the liver, fat and muscle. These results suggest that the Leu452His mutation caused LPL dysfunction and gene transfer of MLPL in vivo produced resistance to the AMPK/PGC-1α signaling pathway in mice.

  11. Catalog of gene expression in adult neural stem cells and their in vivo microenvironment

    International Nuclear Information System (INIS)

    Williams, Cecilia; Wirta, Valtteri; Meletis, Konstantinos; Wikstroem, Lilian; Carlsson, Leif; Frisen, Jonas; Lundeberg, Joakim

    2006-01-01

    Stem cells generally reside in a stem cell microenvironment, where cues for self-renewal and differentiation are present. However, the genetic program underlying stem cell proliferation and multipotency is poorly understood. Transcriptome analysis of stem cells and their in vivo microenvironment is one way of uncovering the unique stemness properties and provides a framework for the elucidation of stem cell function. Here, we characterize the gene expression profile of the in vivo neural stem cell microenvironment in the lateral ventricle wall of adult mouse brain and of in vitro proliferating neural stem cells. We have also analyzed an Lhx2-expressing hematopoietic-stem-cell-like cell line in order to define the transcriptome of a well-characterized and pure cell population with stem cell characteristics. We report the generation, assembly and annotation of 50,792 high-quality 5'-end expressed sequence tag sequences. We further describe a shared expression of 1065 transcripts by all three stem cell libraries and a large overlap with previously published gene expression signatures for neural stem/progenitor cells and other multipotent stem cells. The sequences and cDNA clones obtained within this framework provide a comprehensive resource for the analysis of genes in adult stem cells that can accelerate future stem cell research

  12. Cytomegalovirus replicon-based regulation of gene expression in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Hermine Mohr

    Full Text Available There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV origin of lytic replication (oriLyt, were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.

  13. In vivo protein-DNA interactions at the β-globin gene locus

    International Nuclear Information System (INIS)

    Tohru Ikuta; Yuet Wai Kan

    1991-01-01

    The authors have investigated in vivo protein-DNA interactions in the β-globin gene locus by dimethyl sulfate (DMS) footprinting in K562 cells, which express var-epsilon- and γ-globin but not β-globin. In the locus control region, hypersensitive site 2 (HS-2) exhibited footprints in several putative protein binding motifs. HS-3 was not footprinted. The β promoter was also not footprinted, while extensive footprints were observed in the promoter of the active γ-globin gene. No footprints were seen in the A γ and β3' enhancers. With several motifs, additional protein interactions and alterations in binding patterns occurred with hemin induction. In HeLa cells, some footprints were observed in some of the motifs in HS-2, compatible with the finding that HS-2 has some enhancer function in HeLa cells, albeit much weaker than its activity in K562 cells. No footprint was seen in B lymphocytes. In vivo footprinting is a useful method for studying relevant protein-DNA interactions in erythroid cells

  14. In vivo RNAi screen reveals neddylation genes as novel regulators of Hedgehog signaling.

    Directory of Open Access Journals (Sweden)

    Juan Du

    Full Text Available Hedgehog (Hh signaling is highly conserved in all metazoan animals and plays critical roles in many developmental processes. Dysregulation of the Hh signaling cascade has been implicated in many diseases, including cancer. Although key components of the Hh pathway have been identified, significant gaps remain in our understanding of the regulation of individual Hh signaling molecules. Here, we report the identification of novel regulators of the Hh pathway, obtained from an in vivo RNA interference (RNAi screen in Drosophila. By selectively targeting critical genes functioning in post-translational modification systems utilizing ubiquitin (Ub and Ub-like proteins, we identify two novel genes (dUba3 and dUbc12 that negatively regulate Hh signaling activity. We provide in vivo and in vitro evidence illustrating that dUba3 and dUbc12 are essential components of the neddylation pathway; they function in an enzyme cascade to conjugate the ubiquitin-like NEDD8 modifier to Cullin proteins. Neddylation activates the Cullin-containing ubiquitin ligase complex, which in turn promotes the degradation of Cubitus interruptus (Ci, the downstream transcription factor of the Hh pathway. Our study reveals a conserved molecular mechanism of the neddylation pathway in Drosophila and sheds light on the complex post-translational regulations in Hh signaling.

  15. In vivo tyrosinase mini-gene transfer enhances killing effect of BNCT on amelanotic melanoma

    International Nuclear Information System (INIS)

    Kondoh, H.; Mishima, Y.; Hiratsuka, J.; Iwakura, M.

    2000-01-01

    Using accentuated melanogenesis principally occurring within melanoma cells, we have successfully treated human malignant melanoma (Mm) with 10 B-BPA BNCT. Despite this success, there are still remaining issues for poorly melanogenic Mm and further non-pigment cell tumors. We found the selective accumulation of 10 B-BPA to Mm is primarily due to the complex formation of BPA and melanin-monomers activity synthesized within Mm cells. Then, we succeeded in transferring the tyrosinase gene into amelanotic to substantially produce melanin monomers. These cells has demonstrated increased boron accumulation and enhanced killing effect of BNCT. Further, transfection of TRP-2 (DOPAchrome tautomerase) gene into poorly eumelanotic and slightly phenomelanotic Mm cells in culture cell systems also led to increased BPA accumulation. Thereafter, we studied in vivo gene transfer. We transferred the tyrosinase mini-gene by intra-tumor injection into poorly melanotic Mm proliferating subcutaneously in hamster skin, and performed BNCT. Compared to control tumors, gene-transferred tumors showed increased BPA accumulation leading to enhanced killing effect. (author)

  16. In vivo tyrosinase mini-gene transfer enhances killing effect of BNCT on amelanotic melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Kondoh, H.; Mishima, Y. [Mishima Institute for Dermatological Research, Kobe, Hyogo (Japan); Hiratsuka, J. [Kawasaki Medical School, Dept. of Radiation Oncology, Kurashiki, Okayama (Japan); Iwakura, M. [Kobe Univ. (Japan). School of Medicine

    2000-10-01

    Using accentuated melanogenesis principally occurring within melanoma cells, we have successfully treated human malignant melanoma (Mm) with {sup 10}B-BPA BNCT. Despite this success, there are still remaining issues for poorly melanogenic Mm and further non-pigment cell tumors. We found the selective accumulation of {sup 10}B-BPA to Mm is primarily due to the complex formation of BPA and melanin-monomers activity synthesized within Mm cells. Then, we succeeded in transferring the tyrosinase gene into amelanotic to substantially produce melanin monomers. These cells has demonstrated increased boron accumulation and enhanced killing effect of BNCT. Further, transfection of TRP-2 (DOPAchrome tautomerase) gene into poorly eumelanotic and slightly phenomelanotic Mm cells in culture cell systems also led to increased BPA accumulation. Thereafter, we studied in vivo gene transfer. We transferred the tyrosinase mini-gene by intra-tumor injection into poorly melanotic Mm proliferating subcutaneously in hamster skin, and performed BNCT. Compared to control tumors, gene-transferred tumors showed increased BPA accumulation leading to enhanced killing effect. (author)

  17. Polymeric nanoparticles for nonviral gene therapy extend brain tumor survival in vivo.

    Science.gov (United States)

    Mangraviti, Antonella; Tzeng, Stephany Yi; Kozielski, Kristen Lynn; Wang, Yuan; Jin, Yike; Gullotti, David; Pedone, Mariangela; Buaron, Nitsa; Liu, Ann; Wilson, David R; Hansen, Sarah K; Rodriguez, Fausto J; Gao, Guo-Dong; DiMeco, Francesco; Brem, Henry; Olivi, Alessandro; Tyler, Betty; Green, Jordan J

    2015-02-24

    Biodegradable polymeric nanoparticles have the potential to be safer alternatives to viruses for gene delivery; however, their use has been limited by poor efficacy in vivo. In this work, we synthesize and characterize polymeric gene delivery nanoparticles and evaluate their efficacy for DNA delivery of herpes simplex virus type I thymidine kinase (HSVtk) combined with the prodrug ganciclovir (GCV) in a malignant glioma model. We investigated polymer structure for gene delivery in two rat glioma cell lines, 9L and F98, to discover nanoparticle formulations more effective than the leading commercial reagent Lipofectamine 2000. The lead polymer structure, poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) end-modified with 1-(3-aminopropyl)-4-methylpiperazine, is a poly(β-amino ester) (PBAE) and formed nanoparticles with HSVtk DNA that were 138 ± 4 nm in size and 13 ± 1 mV in zeta potential. These nanoparticles containing HSVtk DNA showed 100% cancer cell killing in vitro in the two glioma cell lines when combined with GCV exposure, while control nanoparticles encoding GFP maintained robust cell viability. For in vivo evaluation, tumor-bearing rats were treated with PBAE/HSVtk infusion via convection-enhanced delivery (CED) in combination with systemic administration of GCV. These treated animals showed a significant benefit in survival (p = 0.0012 vs control). Moreover, following a single CED infusion, labeled PBAE nanoparticles spread completely throughout the tumor. This study highlights a nanomedicine approach that is highly promising for the treatment of malignant glioma.

  18. CRISPR-Cas9 for in vivo Gene Therapy: Promise and Hurdles

    Directory of Open Access Journals (Sweden)

    Wei-Jing Dai

    2016-01-01

    Full Text Available Owing to its easy-to-use and multiplexing nature, the genome editing tool CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR associated nuclease 9 is revolutionizing many areas of medical research and one of the most amazing areas is its gene therapy potentials. Previous explorations into the therapeutic potentials of CRISPR-Cas9 were mainly conducted in vitro or in animal germlines, the translatability of which, however, is either limited (to tissues with adult stem cells amenable to culture and manipulation or currently impermissible (due to ethic concerns. Recently, important progresses have been made on this regard. Several studies have demonstrated the ability of CRISPR-Cas9 for in vivo gene therapy in adult rodent models of human genetic diseases delivered by methods that are potentially translatable to human use. Although these recent advances represent a significant step forward to the eventual application of CRISPR-Cas9 to the clinic, there are still many hurdles to overcome, such as the off-target effects of CRISPR-Cas9, efficacy of homology-directed repair, fitness of edited cells, immunogenicity of therapeutic CRISPR-Cas9 components, as well as efficiency, specificity, and translatability of in vivo delivery methods. In this article, we introduce the mechanisms and merits of CRISPR-Cas9 in genome editing, briefly retrospect the applications of CRISPR-Cas9 in gene therapy explorations and highlight recent advances, later we discuss in detail the challenges lying ahead in the way of its translatability, propose possible solutions, and future research directions.

  19. Targeted decorin gene therapy delivered with adeno-associated virus effectively retards corneal neovascularization in vivo.

    Directory of Open Access Journals (Sweden)

    Rajiv R Mohan

    Full Text Available Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5 impedes corneal neovascularization (CNV in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 µl; 5×10(12 vg/ml application onto the stroma for two minutes after removing corneal epithelium. The levels of CNV were examined with stereomicroscopy, H&E staining, lectin, collagen type IV, CD31 immunocytochemistry and CD31 immunoblotting. Real-time PCR quantified mRNA expression of pro- and anti-angiogenic genes. Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations. Selective decorin delivery into stroma showed significant 52% (p<0.05, 66% (p<0.001, and 63% (p<0.01 reduction at early (day 5, mid (day 10, and late (day 14 stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered corneas in morphometric analysis. The H&E staining, lectin, collagen type IV, CD31 immunostaining (57-65, p<0.5, and CD31 immunoblotting (62-67%, p<0.05 supported morphometric findings. Quantitative PCR studies demonstrated decorin gene therapy down-regulated expression of VEGF, MCP1 and angiopoietin (pro-angiogenic and up-regulated PEDF (anti-angiogenic genes. The clinical, biomicroscopy and transmission electron microscopy studies revealed that AAV5-mediated decorin gene therapy is safe for the cornea. Tissue-targeted AAV5-mediated decorin gene therapy decreases CNV with no major side effects, and could potentially be used for treating patients.

  20. In Vivo Anti-estrogenic Effects of Menadione on Hepatic Estrogen-responsive Gene Expression in Male Medaka (Oryzias latipes)

    OpenAIRE

    Yamaguchi, Akemi; Kohra, Shinya; Ishibashi, Hiroshi; Arizono, Koji; Tominaga, Nobuaki

    2008-01-01

    Menadione, a synthetic vitamin K3, exhibits anti-estrogenic activity on in vitro assay. However, the in vivo anti-estrogenic effects of menadione have not been determined, while correlations between biological effects and structural changes are unclear. Thus, we investigated the in vivo anti-estrogenic activity of menadione under fluorescent light and dark conditions. Suppression of the hepatic estrogen response genes vitellogenin1 (VTG1), VTG2 and estrogen receptor-α (ER-α) was used as an in...

  1. Efficacy of In Vivo Electroporation-Mediated IL-10 Gene Delivery on Survival of Skin Flaps.

    Science.gov (United States)

    Seyed Jafari, S Morteza; Shafighi, Maziar; Beltraminelli, Helmut; Weber, Benedikt; Schmid, Ralph A; Geiser, Thomas; Gazdhar, Amiq; Hunger, Robert E

    2018-04-01

    Despite advances in understanding the underlying mechanisms of flap necrosis and improvement in surgical techniques, skin flap necrosis after reconstructive surgery remains a crucial issue. We investigated the efficacy of electroporation-mediated IL-10 gene transfer to random skin flap with an aim to accelerate wound healing and improve skin flap survival. Nine male Wistar rats (300-330 g) were divided in two groups (a) control group (n = 5), only surgery no gene transfer, and (b) experimental group, received electroporation-mediated IL-10 gene transfer 24 h before the surgery as prophylaxis (n = 4). Random skin flap (McFarlane) was performed in both groups. Planimetry, Laser Doppler imaging, and immunohistochemistry were used to evaluate the effect of IL-10 gene transfer between study groups at day 7. Electroporation-mediated IL-10 gene transfer decreased percentage of flap necrosis (p value = 0.0159) and increased cutaneous perfusion compared to the control group (p value = 0.0159). In addition, Spearman's rank correlation showed a significant negative correlation between percentage of flap necrosis and Laser Index (p value = 0.0083, r -0.83, respectively). Furthermore, significantly higher mean CD31 + vessel density was detected in the experimental group compared to the control group (p value = 0.0159). Additionally, semi-quantitative image analysis showed lower inflammatory cell count in experimental group compared to control group (p value = 0.0317). In vivo electroporation-mediated IL-10 gene transfer reduced necrosis, enhanced survival and vascularity in the ischemic skin flap.

  2. In vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression.

    Science.gov (United States)

    Carlin, Sean; Pugachev, Andrei; Sun, Xiaorong; Burke, Sean; Claus, Filip; O'Donoghue, Joseph; Ling, C Clifton; Humm, John L

    2009-10-01

    To characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer (18)F-fluoromisonidazole ((18)F-FMISO). Three tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: (1) parental R3327-AT, (2) positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, (3) R3327-AT/HRE in which the reporter gene was placed under the control of a hypoxia-inducible factor-responsive promoter sequence (HRE). Animals were coadministered a hypoxia-specific marker (pimonidazole) and the reporter gene probe (124)I-2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-iodouracil ((124)I-FIAU) 3 h prior to sacrifice. Statistical analysis of the spatial association between (124)I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis. Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe (18)F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with (124)I-FIAU (3 h before sacrifice) and (18)F-FMISO (2 h before sacrifice). Statistical analysis of the spatial association between (18)F-FMISO and (124)I-FIAU on a pixel-by-pixel basis was performed. Correlation coefficients between (124)I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, -0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia. Correlation coefficients between (18)F-FMISO and (124)I-FIAU uptakes in R3327-AT/HRE tumors were r=0.56, demonstrating good spatial correspondence between the two tracers. We have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of this model for the

  3. In vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression

    International Nuclear Information System (INIS)

    Carlin, Sean; Pugachev, Andrei; Sun Xiaorong; Burke, Sean; Claus, Filip; O'Donoghue, Joseph; Ling, C. Clifton; Humm, John L.

    2009-01-01

    Purpose: To characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer 18 F-fluoromisonidazole ( 18 F-FMISO). Methods: Three tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: (1) parental R3327-AT, (2) positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, (3) R3327-AT/HRE in which the reporter gene was placed under the control of a hypoxia-inducible factor-responsive promoter sequence (HRE). Animals were coadministered a hypoxia-specific marker (pimonidazole) and the reporter gene probe 124 I-2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodouracil ( 124 I-FIAU) 3 h prior to sacrifice. Statistical analysis of the spatial association between 124 I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis. Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe 18 F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with 124 I-FIAU (3 h before sacrifice) and 18 F-FMISO (2 h before sacrifice). Statistical analysis of the spatial association between 18 F-FMISO and 124 I-FIAU on a pixel-by-pixel basis was performed. Results: Correlation coefficients between 124 I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, -0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia. Correlation coefficients between 18 F-FMISO and 124 I-FIAU uptakes in R3327-AT/HRE tumors were r=0.56, demonstrating good spatial correspondence between the two tracers. Conclusions: We have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of

  4. A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo

    Science.gov (United States)

    Lehto, Taavi; Simonson, Oscar E; Mäger, Imre; Ezzat, Kariem; Sork, Helena; Copolovici, Dana-Maria; Viola, Joana R; Zaghloul, Eman M; Lundin, Per; Moreno, Pedro MD; Mäe, Maarja; Oskolkov, Nikita; Suhorutšenko, Julia; Smith, CI Edvard; Andaloussi, Samir EL

    2011-01-01

    Finding suitable nonviral delivery vehicles for nucleic acid–based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice. PMID:21343913

  5. Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma.

    Science.gov (United States)

    Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

    2016-04-01

    Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P leiomyoma lesions with both targeted and untargeted adenovirus. Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. © The Author(s) 2016.

  6. In Vivo Testing of MicroRNA-Mediated Gene Knockdown in Zebrafish

    Directory of Open Access Journals (Sweden)

    Ivone Un San Leong

    2012-01-01

    Full Text Available The zebrafish (Danio rerio has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis. The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.

  7. Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo

    Science.gov (United States)

    White, JD; Thesier, DM; Swain, JBD; Katz, MG; Tomasulo, C; Henderson, A; Wang, L; Yarnall, C; Fargnoli, A; Sumaroka, M; Isidro, A; Petrov, M; Holt, D; Nolen-Walston, R; Koch, WJ; Stedman, HH; Rabinowitz, J; Bridges, CR

    2013-01-01

    We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy. PMID:21228882

  8. Cell-type-specific gene delivery into neuronal cells in vitro and in vivo

    International Nuclear Information System (INIS)

    Parveen, Zahida; Mukhtar, Muhammad; Rafi, Mohammed; Wenger, David A.; Siddiqui, Khwaja M.; Siler, Catherine A.; Dietzschold, Bernhard; Pomerantz, Roger J.; Schnell, Matthias J.; Dornburg, Ralph

    2003-01-01

    The avian retroviruses reticuloendotheliosis virus strain A (REV-A) and spleen necrosis virus (SNV) are not naturally infectious in human cells. However, REV-A-derived viral vectors efficiently infect human cells when they are pseudotyped with envelope proteins displaying targeting ligands specific for human cell-surface receptors. Here we report that vectors containing the gag region of REV-A and pol of SNV can be pseudotyped with the envelope protein of vesicular stomatitis virus (VSV) and the glycoproteins of different rabies virus (RV) strains. Vectors pseudotyped with the envelope protein of the highly neurotropic RV strain CVS-N2c facilitated cell type-specific gene delivery into mouse and human neurons, but did not infect other human cell types. Moreover, when such vector particles were injected into the brain of newborn mice, only neuronal cells were infected in vivo. Cell-type-specific gene delivery into neurons may present quite specific gene therapy approaches for many degenerative diseases of the brain

  9. The prognostic value of temporal in vitro and in vivo derived hypoxia gene-expression signatures in breast cancer

    International Nuclear Information System (INIS)

    Starmans, Maud H.W.; Chu, Kenneth C.; Haider, Syed; Nguyen, Francis; Seigneuric, Renaud; Magagnin, Michael G.; Koritzinsky, Marianne; Kasprzyk, Arek; Boutros, Paul C.; Wouters, Bradly G.

    2012-01-01

    Background and purpose: Recent data suggest that in vitro and in vivo derived hypoxia gene-expression signatures have prognostic power in breast and possibly other cancers. However, both tumour hypoxia and the biological adaptation to this stress are highly dynamic. Assessment of time-dependent gene-expression changes in response to hypoxia may thus provide additional biological insights and assist in predicting the impact of hypoxia on patient prognosis. Materials and methods: Transcriptome profiling was performed for three cell lines derived from diverse tumour-types after hypoxic exposure at eight time-points, which include a normoxic time-point. Time-dependent sets of co-regulated genes were identified from these data. Subsequently, gene ontology (GO) and pathway analyses were performed. The prognostic power of these novel signatures was assessed in parallel with previous in vitro and in vivo derived hypoxia signatures in a large breast cancer microarray meta-dataset (n = 2312). Results: We identified seven recurrent temporal and two general hypoxia signatures. GO and pathway analyses revealed regulation of both common and unique underlying biological processes within these signatures. None of the new or previously published in vitro signatures consisting of hypoxia-induced genes were prognostic in the large breast cancer dataset. In contrast, signatures of repressed genes, as well as the in vivo derived signatures of hypoxia-induced genes showed clear prognostic power. Conclusions: Only a subset of hypoxia-induced genes in vitro demonstrates prognostic value when evaluated in a large clinical dataset. Despite clear evidence of temporal patterns of gene-expression in vitro, the subset of prognostic hypoxia regulated genes cannot be identified based on temporal pattern alone. In vivo derived signatures appear to identify the prognostic hypoxia induced genes. The prognostic value of hypoxia-repressed genes is likely a surrogate for the known importance of

  10. AAV2-mediated in vivo immune gene therapy of solid tumours

    LENUS (Irish Health Repository)

    Collins, Sara A

    2010-12-20

    Abstract Background Many strategies have been adopted to unleash the potential of gene therapy for cancer, involving a wide range of therapeutic genes delivered by various methods. Immune therapy has become one of the major strategies adopted for cancer gene therapy and seeks to stimulate the immune system to target tumour antigens. In this study, the feasibility of AAV2 mediated immunotherapy of growing tumours was examined, in isolation and combined with anti-angiogenic therapy. Methods Immune-competent Balb\\/C or C57 mice bearing subcutaneous JBS fibrosarcoma or Lewis Lung Carcinoma (LLC) tumour xenografts respectively were treated by intra-tumoural administration of AAV2 vector encoding the immune up-regulating cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) and the co-stimulatory molecule B7-1 to subcutaneous tumours, either alone or in combination with intra-muscular (IM) delivery of AAV2 vector encoding Nk4 14 days prior to tumour induction. Tumour growth and survival was monitored for all animals. Cured animals were re-challenged with tumourigenic doses of the original tumour type. In vivo cytotoxicity assays were used to investigate establishment of cell-mediated responses in treated animals. Results AAV2-mediated GM-CSF, B7-1 treatment resulted in a significant reduction in tumour growth and an increase in survival in both tumour models. Cured animals were resistant to re-challenge, and induction of T cell mediated anti-tumour responses were demonstrated. Adoptive transfer of splenocytes to naïve animals prevented tumour establishment. Systemic production of Nk4 induced by intra-muscular (IM) delivery of Nk4 significantly reduced subcutaneous tumour growth. However, combination of Nk4 treatment with GM-CSF, B7-1 therapy reduced the efficacy of the immune therapy. Conclusions Overall, this study demonstrates the potential for in vivo AAV2 mediated immune gene therapy, and provides data on the inter-relationship between tumour

  11. glpx Gene in Mycobacterium tuberculosis Is Required for In Vitro Gluconeogenic Growth and In Vivo Survival.

    Directory of Open Access Journals (Sweden)

    Hiten J Gutka

    Full Text Available Several enzymes involved in central carbon metabolism and gluconeogenesis play a critical role in survival and pathogenesis of Mycobacterium tuberculosis (Mtb. The only known functional fructose 1,6-bisphosphatase (FBPase in Mtb is encoded by the glpX gene and belongs to the Class II sub-family of FBPase. We describe herein the generation of a ΔglpX strain using homologous recombination. Although the growth profile of ΔglpX is comparable to that of wild type Mtb when grown on the standard enrichment media, its growth is dysgonic with individual gluconeogenic substrates such as oleic acid, glycerol and acetate. In mice lung CFU titers of ΔglpX were 2-3 log10 lower than the wild-type Mtb strain. The results indicate that glpX gene encodes a functional FBPase and is essential for both in vitro and in vivo growth and survival of Mtb. Loss of glpX results in significant reduction of FBPase activity but not complete abolition. These findings verify that the glpX encoded FBPase II in Mtb can be a potential target for drug discovery.

  12. Gene gun bombardment-mediated expression and translocation of EGFP-tagged GLUT4 in skeletal muscle fibres in vivo

    DEFF Research Database (Denmark)

    Lauritzen, Hans P M M; Reynet, Christine; Schjerling, Peter

    2002-01-01

    the enhanced green fluorescent protein (EGFP) labelling technique with physical transfection methods in vivo: intramuscular plasmid injection or gene gun bombardment. During optimisation experiments with plasmid coding for the EGFP reporter alone EGFP-positive muscle fibres were counted after collagenase...... treatment of in vivo transfected flexor digitorum brevis (FDB) muscles. In contrast to gene gun bombardment, intramuscular injection produced EGFP expression in only a few fibres. Regardless of the transfection technique, EGFP expression was higher in muscles from 2-week-old rats than in those from 6-week......Cellular protein trafficking has been studied to date only in vitro or with techniques that are invasive and have a low time resolution. To establish a gentle method for analysis of glucose transporter-4 (GLUT4) trafficking in vivo in fully differentiated rat skeletal muscle fibres we combined...

  13. A molecular toolbox for rapid generation of viral vectors to up- or down-regulate in vivo neuronal gene expression

    Directory of Open Access Journals (Sweden)

    Melanie D. White

    2011-07-01

    Full Text Available We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1 and Kir3.2 and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miR. We show that AAV assembled to express HCN1 miR produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miR with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience.

  14. Bifidobacterium breve as a delivery vector of IL-24 gene therapy for head and neck squamous cell carcinoma in vivo.

    Science.gov (United States)

    Wang, L; Vuletic, I; Deng, D; Crielaard, W; Xie, Z; Zhou, K; Zhang, J; Sun, H; Ren, Q; Guo, C

    2017-11-01

    Beneficial bacteria are becoming ever more popular gene delivery method for hypoxia-tumor targeting in vivo. In this study we investigated the therapeutic effect of new recombinant Bifidobacterium breve strain expressing interleukin (IL)-24 gene (B. breve-IL24) on head and neck tumor xenograft in mice. Briefly, B. breve transformants were obtained through electro-transformation. Bacteria-tumor-targeting ability were analyzed in vivo over different time points (1, 3 and 7 days post-bacteria injection). Furthermore, the therapeutic effect of bacteria on tumor cells in vivo were analyzed as follows: 30 Balb/c nude mice bearing subcutaneous tumor were randomly divided in three groups (Drug group, green fluorescent protein (GFP) group and Saline group). The therapy lasted for 2 weeks and included B. breve-IL24 administration via tail vein for Drug group, B. breve-GFP for GFP group and phosphate buffered saline for Saline group. The tumor growth was monitored using standard caliper technique, while the apoptosis induction in vivo was analyzed by Real-time Positron Emission Tomography/Computed Tomography (PET/CT) imaging ([18F]-ML-10 tracer). At the end of the experiment, tumor tissues were collected and analyzed by western blotting. Briefly, our results suggested that our new recombinant bacterium has the capability of targeting tumor tissue in vivo. As for the therapeutic effect, our new strain has revealed to be a promising therapeutic approach against tumor growth in vivo. Briefly, higher tumor growth inhibition and higher tumor cell apoptosis induction were observed in Drug group compared with the GFP and Saline groups. To conclude, a new recombinant strain B. breve-IL24 offers a novel, safe and clinically acceptable therapeutic approach for tumor therapy in vivo.

  15. Effect of irradiation on gene expression of rat liver adhesion molecules. In vivo and in vitro studies

    International Nuclear Information System (INIS)

    Moriconi, Federico; Malik, Ihtzaz; Ahmad, Ghayyor; Dudas, Joszef; Ramadori, Giuliano; Rave-Fraenk, Margret; Vorwerk, Hilke; Hille, Andrea; Hess, Clemens Friedrich; Christiansen, Hans

    2009-01-01

    Background and purpose: Migration of leukocytes into tissue is a key element of innate and adaptive immunity. An animal study showed that liver irradiation, in spite of induction of chemokine gene expression, does not lead to recruitment of leukocytes into the parenchyma. The aim of this study was to analyze gene expression of adhesion molecules, which mediate leukocyte recruitment into organs, in irradiated rat liver in vivo and rat hepatocytes in vitro. Material and methods: Rat livers in vivo were irradiated selectively at 25 Gy. Isolated hepatocytes in vitro were irradiated at 8 Gy. RNA extracted within 48 h after irradiation in vivo and in vitro was analyzed by real-time PCR (polymerase chain reaction) and Northern blot. Adhesion molecule concentration in serum was measured by ELISA (enzyme-linked immunosorbent assay). Cryostat sections of livers were used for immunohistology. Results: Significant radiation-induced increase of ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), JAM-1 (junctional adhesion molecule-1), β 1 -integrin, β 2 -integrin, E-cadherin, and P-selectin gene expression could be detected in vivo, while PECAM-1 (platelet-endothelial cell adhesion molecule-1) gene expression remained unchanged. In vitro, β 1 -integrin, JAM-1, and ICAM-2 showed a radiation-induced increased expression, whereas the levels of P-selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 (mucosal addressin cell adhesion molecule-1), β 2 -integrin, and E-cadherin were downregulated. However, incubation of irradiated hepatocytes with either tumor necrosis factor-(TNF-)α, interleukin-(IL-)1β, or IL-6 plus TNF-α led to an upregulation of P-selectin, ICAM-1 and VCAM-1. Conclusion: The findings suggest that liver irradiation modulates gene expression of the main adhesion molecules in vivo and in cytokine-activated hepatocytes, with the exception of PECAM-1. This may be one reason for the lack of inflammation in the irradiated rat liver. (orig.)

  16. X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts.

    Directory of Open Access Journals (Sweden)

    Chi-Hun Park

    Full Text Available To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF, and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process

  17. Synthesis and evaluation of cationic nanomicelles for in vitro and in vivo gene delivery

    Science.gov (United States)

    Mandke, Rhishikesh Subhash

    The goal of proposed study was to contribute towards the development of a nano size, high efficiency and low toxicity non-viral polymeric vector for gene delivery in vitro and in vivo. A series of fatty acid grafted low-molecular-weight chitosan (N-acyl LMWCs) were synthesized, purified and characterized for their physicochemical properties using various analytical techniques such as infrared spectroscopy, elemental analysis and dynamic light scattering. The formulation parameters including pH, sonication duration, and filtration altered the physicochemical characteristics of N-acyl LMWC nanomicelles. The acyl chain length and degree of unsaturation in fatty acids also had an impact on the physicochemical properties and the transfection efficiency of nanomicelles. N-acyl LMWC nanomicelles showed efficient in vitro transfection as visualized and quantified using a reporter plasmid (encoding green fluorescent protein), and therapeutic plasmids (encoding for interleukin-4 and interleukin-10), respectively. The in vitro transfection efficiencies of N-acyl LMWCs with 18:1 and 18:2 grafts (oleic and linoleic acids) were comparable with FuGENERTM HD (marketed non-viral vector) but were ˜8-fold and 35-fold higher as compared to LMWC and naked DNA, respectively. The in vivo transfection efficiency of N-acyl LMWC to deliver plasmids individually encoding IL-4 and IL-10 as well as a bicistronic plasmid encoding both IL-4 and IL-10 was studied in a multiple, low-dose streptozotocin induced diabetic mouse model. The transfection efficiency of pDNA/N-acyl LMWC polyplexes injected via intramuscular route showed significant improvement (ptumor necrosis factor-alpha (TNF-alpha) levels. The pancreas of pDNA/N-acyl LMWC polyplex treated animals exhibited protection from streptozotocin-induced insulitis and the delivery systems were biocompatible. Histological studies revealed that there were no signs of chronic inflammation at the injection site. The bicistronic plasmid exhibited

  18. UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene

    Directory of Open Access Journals (Sweden)

    M. Zeeshan Chaudhry

    2017-07-01

    Full Text Available Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV UL36 gene, pUL36 (also known as vICA, binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV homolog of the UL36 gene is called M36, and its protein product (pM36 is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMVUL36 inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo.

  19. Utility of in vivo transcription profiling for identifying Pseudomonas aeruginosa genes needed for gastrointestinal colonization and dissemination

    DEFF Research Database (Denmark)

    Koh, Andrew Y; Mikkelsen, Per J; Smith, Roger S

    2010-01-01

    these mutants and WT P. aeruginosa PA14. To evaluate T3SS factors, we tested GI colonization and neutropenia-induced dissemination of both deletional (PAO1 and PAK) and insertional (PA14) mutants in four genes in the P. aeruginosa T3SS, exoS or exoU, exoT, and popB. There were no significant differences in GI......, increased transcription of genes during in vivo murine GI colonization is not predictive of an essential role for the gene product in either colonization or overall survival following induction of neutropenia....

  20. Rapid In Vivo Validation of Tumor Suppressor Gene Function in Prostate Cancer Progression

    Science.gov (United States)

    2016-07-01

    identification of the best sgRNA sequences and accelerated our ability to move to the in vivo studies proposed in Aim2. Our goal was to use CRISPR / Cas ...and to initiate prostate cancer in the mouse after injection of lentiviral particles expressing CRISPR / Cas components and Cre recombinase. Our initial...in vivo Our goal was to use CRISPR / Cas lentiviral transduction of the adult prostate to inactivate p53 or Rb. We aimed to recapitulate the effects of

  1. Retroviral packaging cells encapsulated in TheraCyte immunoisolation devices enable long-term in vivo gene delivery.

    Science.gov (United States)

    Krupetsky, Anna; Parveen, Zahida; Marusich, Elena; Goodrich, Adrienne; Dornburg, Ralph

    2003-05-01

    The method of delivering a therapeutic gene into a patient is still one of the major obstacles towards successful human gene therapy. Here we describe a novel gene delivery approach using TheraCyte immunoisolation devices. Retroviral vector producing cells, derived from the avian retrovirus spleen necrosis virus, SNV, were encapsulated in TheraCyte devices and tested for the release of retroviral vectors. In vitro experiments show that such devices release infectious retroviral vectors into the tissue culture medium for up to 4 months. When such devices were implanted subcutaneously in SCID mice, infectious virus was released into the blood stream. There, the vectors were transported to and infected tumors, which had been induced by subcutaneous injection of tissue culture cells. Thus, this novel concept of a continuous, long-term gene delivery may constitute an attractive approach for future in vivo human gene therapy.

  2. Efficient in vivo gene transfer to xenotransplanted human skin by lentivirus-mediated, but not by AAV-directed, gene delivery

    DEFF Research Database (Denmark)

    Jakobsen, Maria Vad; Askou, Anne Louise; Dokkedahl, Karin Stenderup

    skin graft, and firefly luciferase expression was observed primarily in neighboring tissue beneath or surrounding the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin...... graft only. The study demonstrates limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo....

  3. Class I gene regulation of haplotype preference may influence antiviral immunity in vivo

    DEFF Research Database (Denmark)

    Thomsen, Allan Randrup; Marker, O

    1989-01-01

    targets. In regard to the in vivo significance of haplotype preference it was found that (C X C3) F1 mice expressed an earlier and stronger virus-specific delayed type hypersensitivity response and exerted a more efficient virus control than did (C-H-2dm2 X C3) F1. Taken together these findings suggest...... that haplotype preference reflects a selection process favoring the restriction element associated with the most efficient immune response in vivo. The implications of this are discussed....

  4. Gene expression and apoptosis in bovine embryos during in vitro culture and in vivo development

    NARCIS (Netherlands)

    Knijn, H.W.

    2004-01-01

    The first attempts to fertilise in vitro bovine oocytes were done in the late sixties but only in 1982 the first calf was born after transplantation of a complete in vitro produced embryo. Since then the in vitro production system improved a lot but it is still impossible to mimic the in vivo

  5. Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Madsen, O.D.; Andersen, L.C.; Michelsen, B.; Owerbach, D.; Larsson, L.I.; Lernmark, A.; Steiner, D.F. (Hagedorn Research Laboratory, Gentofte (Denmark))

    1988-09-01

    The pluripotent rat islet tumor cell line MSL-G2 expresses primarily glucagon or cholecystokinin and not insulin in vitro but changes phenotype completely after prolonged in vivo cultivation to yield small-sized hypoglycemic tumors composed almost entirely of insulin-producing beta cells. When a genomic DNA fragment containing the coding and upstream regulatory regions of the human insulin gene was stably transfected into MSL-G2 cells no measurable amounts of insulin or insulin mRNA were detected in vitro. However, successive transplantation of two transfected clones resulted in hypoglycemic tumors that efficiently coexpressed human and rat insulin as determined by human C-peptide-specific immunoreagents. These results demonstrate that cis-acting tissue-specific insulin gene enhancer elements are conserved between rat and human insulin genes. The authors propose that the in vivo differentiation of MSL-G2 cells and transfected subclones into insulin-producing cells reflects processes of natural beta-cell ontogeny leading to insulin gene expression.

  6. Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo

    International Nuclear Information System (INIS)

    Madsen, O.D.; Andersen, L.C.; Michelsen, B.; Owerbach, D.; Larsson, L.I.; Lernmark, A.; Steiner, D.F.

    1988-01-01

    The pluripotent rat islet tumor cell line MSL-G2 expresses primarily glucagon or cholecystokinin and not insulin in vitro but changes phenotype completely after prolonged in vivo cultivation to yield small-sized hypoglycemic tumors composed almost entirely of insulin-producing beta cells. When a genomic DNA fragment containing the coding and upstream regulatory regions of the human insulin gene was stably transfected into MSL-G2 cells no measurable amounts of insulin or insulin mRNA were detected in vitro. However, successive transplantation of two transfected clones resulted in hypoglycemic tumors that efficiently coexpressed human and rat insulin as determined by human C-peptide-specific immunoreagents. These results demonstrate that cis-acting tissue-specific insulin gene enhancer elements are conserved between rat and human insulin genes. The authors propose that the in vivo differentiation of MSL-G2 cells and transfected subclones into insulin-producing cells reflects processes of natural beta-cell ontogeny leading to insulin gene expression

  7. Targeted in vitro and in vivo gene transfer into T Lymphocytes: potential of direct inhibition of allo-immune activation

    Directory of Open Access Journals (Sweden)

    Mehra Mandeep R

    2006-11-01

    Full Text Available Abstract Background Successful inhibition of alloimmune activation in organ transplantation remains one of the key events in achieving a long-term graft survival. Since T lymphocytes are largely responsible for alloimmune activation, targeted gene transfer of gene of cyclin kinase inhibitor p21 into T cells might inhibit their aberrant proliferation. A number of strategies using either adenoviral or lentiviral vectors linked to mono or bispecific antibodies directed against T cell surface markers/cytokines did not yield the desired results. Therefore, this study was designed to test if a CD3promoter-p21 chimeric construct would in vitro and in vivo transfer p21 gene to T lymphocytes and result in inhibition of proliferation. CD3 promoter-p21 chimeric constructs were prepared with p21 in the sense and antisense orientation. For in vitro studies EL4-IL-2 thyoma cells were used and for in vivo studies CD3p21 sense and antisense plasmid DNA was injected intramuscularly in mice. Lymphocyte proliferation was quantified by 3H-thymidine uptake assay; IL-2 mRNA expression was studied by RT-PCR and using Real Time PCR assay, we monitored the CD3, p21, TNF-α and IFN-γ mRNA expression. Results Transfection of CD3p21 sense and antisense in mouse thyoma cell line (EL4-IL-2 resulted in modulation of mitogen-induced proliferation. The intramuscular injection of CD3p21 sense and antisense plasmid DNA into mice also modulated lymphocyte proliferation and mRNA expression of pro-inflammatory cytokines. Conclusion These results demonstrate a novel strategy of in vitro and in vivo transfer of p21 gene to T cells using CD3-promoter to achieve targeted inhibition of lymphocyte proliferation and immune activation.

  8. Role of gene 59 of bacteriophage T4 in repair of uv-irradiated and alkylated DNA in vivo

    International Nuclear Information System (INIS)

    Wu, R.; Wu, J.L.; Yeh, Y.C.

    1975-01-01

    Nonsense mutants in gene 59 (amC5, am HL628) were used to study the role of this gene in the repair of uv-damaged and alkylated DNA of bacteriophage T4 in vivo. The higher sensitivity to uv irradiation and alkylation of gene 59 mutants after exposure to these agents was established by a comparison of the survival fractions with wild type. Zonal centrifugal analysis of both parental and nascent mutant intracellular DNA molecules after uv irradiation showed that immediately after exposure the size of single-stranded DNA fragments was the same as the wild-type intracellular DNA. However, the capability of rejoining fragmented intracellular DNA was greatly reduced in the mutant. In contrast, the wild-type-infected cells under the same condition resumed DNA replication and repaired its DNA to normal size. Methyl methanesulfonate induced more randomly fragmented intracellular DNA, when compared to uv irradiation. The rate of rejoining under these conditions as judged from their sedimentation profiles was also greatly reduced in mutant-infected cells. Further evidence is presented that uv repair is not a simple consequence of arrested DNA replication, which is a phenotype of the mutant when infected in a nonpermissive host, Escherichia coli B(su - ), but rather that the DNA repair function of gene 59 is independent of the replication function. These and other data presented indicate that a product(s) of gene 59 is essential for both repair of uv lesions and repair of alkylation damage of DNA in vivo. It is suggested that gene 59 may have two functions during viral development: DNA replication and replication repair of DNA molecules

  9. Regulatory elements in vivo in the promoter of the abscisic acid responsive gene rab17 from maize.

    Science.gov (United States)

    Busk, P K; Jensen, A B; Pagès, M

    1997-06-01

    The rab17 gene from maize is transcribed in late embryonic development and is responsive to abscisic acid and water stress in embryo and vegetative tissues. In vivo footprinting and transient transformation of rab17 were performed in embryos and vegetative tissues to characterize the cis-elements involved in regulation of the gene. By in vivo footprinting, protein binding was observed to nine elements in the promoter, which correspond to five putative ABREs (abscisic acid responsive elements) and four other sequences. The footprints indicated that distinct proteins interact with these elements in the two developmental stages. In transient transformation, six of the elements were important for high level expression of the rab17 promoter in embryos, whereas only three elements were important in leaves. The cis-acting sequences can be divided in embryo-specific, ABA-specific and leaf-specific elements on the basis of protein binding and the ability to confer expression of rab17. We found one positive, new element, called GRA, with the sequence CACTGGCCGCCC. This element was important for transcription in leaves but not in embryos. Two other non-ABRE elements that stimulated transcription from the rab17 promoter resemble previously described abscisic acid and drought-inducible elements. There were differences in protein binding and function of the five ABREs in the rab17 promoter. The possible reasons for these differences are discussed. The in vivo data obtained suggest that an embryo-specific pathway regulates transcription of the rab genes during development, whereas another pathway is responsible for induction in response to ABA and drought in vegetative tissues.

  10. Optical imaging of luminescence for in vivo quantification of gene electrotransfer in mouse muscle and knee

    Directory of Open Access Journals (Sweden)

    Scherman D

    2006-03-01

    Full Text Available Abstract Background Optical imaging is an attractive non-invasive way to evaluate the expression of a transferred DNA, mainly thanks to its lower cost and ease of realization. In this study optical imaging was evaluated for monitoring and quantification of the mouse knee joint and tibial cranial muscle electrotransfer of a luciferase encoding plasmid. Optical imaging was applied to study the kinetics of luciferase expression in both tissues. Results The substrate of luciferase (luciferin was injected either intraperitonealy (i.p. or in situ into the muscle or the knee joint. Luminescence resulting from the luciferase-luciferin reaction was measured in vivo with a cooled CCD camera and/or in vitro on tissue lysate. Maximal luminescence of the knee joint and muscle after i.p. (2.5 mg or local injection of luciferin (50 μg in the knee joint, 100 μg in the muscle were highly correlated. With the local injection procedure adopted, in vivo and in vitro luminescences measured on the same muscles significantly correlated. Luminescence measurements were reproducible and the signal level was proportional to the amount of plasmid injected. In vivo luciferase activity in the electrotransfered knee joint was detected for two weeks. Intramuscular electrotransfer of 0.3 or 3 μg of plasmid led to stable luciferase expression for 62 days, whereas injecting 30 μg of plasmid resulted in a drop of luminescence three weeks after electrotransfer. These decreases were partially associated with the development of an immune response. Conclusion A particular advantage of the i.p. injection of substrate is a widespread distribution at luciferase production sites. We have also highlighted advantages of local injection as a more sensitive detection method with reduced substrate consumption. Besides, this route of injection is relatively free of uncontrolled parameters, such as diffusion to the target organ, crossing of biological barriers and evidencing variations in

  11. AAV-Mediated Gene Targeting Is Significantly Enhanced by Transient Inhibition of Nonhomologous End Joining or the Proteasome In Vivo

    Science.gov (United States)

    Paulk, Nicole K.; Loza, Laura Marquez; Finegold, Milton J.

    2012-01-01

    Abstract Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah−/−Ku70−/− double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy. PMID:22486314

  12. Tissue-specific expression of silkmoth chorion genes in vivo using Bombyx mori nuclear polyhedrosis virus as a transducing vector.

    Science.gov (United States)

    Iatrou, K; Meidinger, R G

    1990-01-01

    A pair of silkmoth chorion chromosomal genes, HcA.12-HcB.12, was inserted into a baculovirus transfer vector, pBmp2, derived from the nuclear polyhedrosis virus of Bombyx mori. This vector, which permits the insertion of foreign genetic material in the vicinity of a mutationally inactivated polyhedrin gene, was used to acquire the corresponding recombinant virus. Injection of mutant silkmoth pupae that lack all Hc chorion genes with the recombinant virus resulted in the infection of all internal organs including follicular tissue. Analysis of RNA from infected tissues has demonstrated that the two chorion genes present in the viral genome are correctly transcribed under the control of their own promoter in follicular cells, the tissue in which chorion genes are normally expressed. The chorion primary transcripts are also correctly processed in the infected follicular cells and yield mature mRNAs indistinguishable from authentic chorion mRNAs present in wild-type follicles. These results demonstrate that recombinant nuclear polyhedrosis viruses can be used as transducing vectors for introducing genetic material of host origin into the cells of the organism and that the transduced genes are transiently expressed in a tissue-specific manner under the control of their resident regulatory sequences. Thus we show the in vivo expression of cloned genes under cellular promoter control in an insect other than Drosophila melanogaster. The approach should be applicable to all insect systems that are subject to nuclear polyhedrosis virus infection. Images PMID:2187186

  13. In vivo site-specific mutagenesis and gene collage using the delitto perfetto system in yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Stuckey, Samantha; Mukherjee, Kuntal; Storici, Francesca

    2011-01-01

    Delitto perfetto is a site-specific in vivo mutagenesis system that has been developed to generate changes at will in the genome of the yeast Saccharomyces cerevisiae. Using this technique, it is possible to rapidly and efficiently engineer yeast strains without requiring several intermediate steps as it functions in only two steps, both of which rely on homologous recombination to drive the changes to the target DNA region. The first step involves the insertion of a cassette containing two markers at or near the locus to be altered. The second step involves complete removal of this cassette with oligonucleotides and/or other genetic material and transfer of the expected genetic modification(s) to the chosen DNA locus. Here we provide a detailed protocol of the delitto perfetto approach and present examples of the most common and useful applications for in vivo mutagenesis to generate base substitutions, deletions, insertions, as well as for precise in vivo assembly and integration of multiple genetic elements, or gene collage.

  14. Changes in cytochrome P450 gene expression and enzyme activity induced by xenobiotics in rabbits in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Orsolya Palócz

    2017-06-01

    Full Text Available As considerable inter-species differences exist in xenobiotic metabolism, developing new pharmaceutical therapies for use in different species is fraught with difficulties. For this reason, very few medicines have been registered for use in rabbits, despite their importance in inter alia meat and fur production. We have developed a rapid and sensitive screening system for drug safety in rabbits based on cytochrome P450 enzyme assays, specifically CYP1A1, CYP1A2 and CYP3A6, employing an adaptation of the luciferin-based clinical assay currently used in human drug screening. Short-term (4-h cultured rabbit primary hepatocytes were treated with a cytochrome inducer (phenobarbital and 2 inhibitors (alpha-naphthoflavone and ketoconazole. In parallel, and to provide verification, New Zealand white rabbits were dosed with 80 mg/kg phenobarbital or 40 mg/kg ketoconazole for 3 d. Ketoconazole significantly increased CYP3A6 gene expression and decreased CYP3A6 activity both in vitro and in vivo. CYP1A1 activity was decreased by ketoconazole in vitro and increased in vivo. This is the first report of the inducer effect of ketoconazole on rabbit cytochrome isoenzymes in vivo. Our data support the use of a luciferin-based assay in short-term primary hepatocytes as an appropriate tool for xenobiotic metabolism assays and short-term toxicity testing in rabbits.

  15. The FBPase Encoding Gene glpX Is Required for Gluconeogenesis, Bacterial Proliferation and Division In Vivo of Mycobacterium marinum.

    Science.gov (United States)

    Tong, Jingfeng; Meng, Lu; Wang, Xinwei; Liu, Lixia; Lyu, Liangdong; Wang, Chuan; Li, Yang; Gao, Qian; Yang, Chen; Niu, Chen

    2016-01-01

    Lipids have been identified as important carbon sources for Mycobacterium tuberculosis (Mtb) to utilize in vivo. Thus gluconeogenesis bears a key role for Mtb to survive and replicate in host. A rate-limiting enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (FBPase) is encoded by the gene glpX. The functions of glpX were studied in M. marinum, a closely related species to Mtb. The glpX deletion strain (ΔglpX) displayed altered gluconeogenesis, attenuated virulence, and altered bacterial proliferation. Metabolic profiles indicate an accumulation of the FBPase substrate, fructose 1, 6-bisphosphate (FBP) and altered gluconeogenic flux when ΔglpX is cultivated in a gluconeogenic carbon substrate, acetate. In both macrophages and zebrafish, the proliferation of ΔglpX was halted, resulting in dramatically attenuated virulence. Intracellular ΔglpX exhibited an elongated morphology, which was also observed when ΔglpX was grown in a gluconeogenic carbon source. This elongated morphology is also supported by the observation of unseparated multi-nucleoid cell, indicating that a complete mycobacterial division in vivo is correlated with intact gluconeogenesis. Together, our results indicate that glpX has essential functions in gluconeogenesis, and plays an indispensable role in bacterial proliferation in vivo and virulence of M. marinum.

  16. In vivo PET imaging with 18F-FHBG of hepatoma cancer gene therapy using herpes simplex virus thymidine kinase and ganciclovir

    International Nuclear Information System (INIS)

    Lee, TaeSup; Kim, JunYoup; Moon, ByungSeok; Kang, JooHyun; Song, Inho; Kwon, HeeChung; Kim, KyungMin; Cheon, GiJeong; Choi, ChangWoon; Lim, SangMoo

    2007-01-01

    Monitoring gene expression in vivo to evaluate the gene therapy efficacy is a critical issue for scientists and physicians. Non-invasive nuclear imaging can offer information regarding the level of gene expression and its location when an appropriate reporter gene is constructed in the therapeutic gene therapy. Herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) is the most common reporter gene and is used in cancer gene therapy by activating relatively nontoxic compounds, such as acyclovir or ganciclovir (GCV), to induce cell death. In this study, we investigate the feasibility of monitoring cancer gene therapy using retroviral vector transduced HSV1-tk and GCV, in vitro cellular uptake and in vivo animal studies, including biodistribution and small animal positron emission tomography (PET) imaging, were performed in HSV1-tk and luciferase (Luc)-transduced MCA-TK/Luc and enhanced green fluorescent protein (eGFP)-transduced MCA-eGFP hepatoma cell lines

  17. In vivo oestrogenic modulation of Egr1 and Pitx1 gene expression in female rat pituitary gland.

    Science.gov (United States)

    Gajewska, Alina; Herman, Andrzej P; Wolińska-Witort, Ewa; Kochman, Kazimierz; Zwierzchowski, Lech

    2014-12-01

    EGR1 and PITX1 are transcription factors required for gonadotroph cell Lhb promoter activation. To determine changes in Egr1 and Pitx1 mRNA levels in central and peripheral pituitary stimulations, an in vivo model based on i.c.v. pulsatile (1 pulse/0.5 h over 2 h) GnRH agonist (1.5 nM buserelin) or antagonist (2 nM antide) microinjections was used. The microinjections were given to ovariectomised and 17β-oestradiol (E2) (3×20 μg), ERA (ESR1) agonist propyl pyrazole triol (PPT) (3×0.5 mg), ERB (ESR2) agonist diarylpropionitrile (DPN) (3×0.5 mg) s.c. pre-treated rats 30 min after last pulse anterior pituitaries were excised. Relative mRNA expression was determined by quantitative RT-PCR (qRT-PCR). Results revealed a gene-specific response for GnRH and/or oestrogenic stimulations in vivo. Buserelin pulses enhanced Egr1 expression by 66% in ovariectomised rats, whereas the oestradiol-supplemented+i.c.v. NaCl-microinjected group showed a 50% increase in Egr1 mRNA expression. The oestrogenic signal was transmitted via ERA (ESR1) and ERB (ESR2) activation as administration of PPT and DPN resulted in 97 and 62%, respectively, elevation in Egr1 mRNA expression. A synergistic action of GnRH agonist and 17β-oestradiol (E2) stimulation of the Egr1 gene transcription in vivo were found. GnRHR activity did not affect Pitx1 mRNA expression; regardless of NaCl, buserelin or antide i.c.v. pulses, s.c. oestrogenic supplementation (with E2, PPT or DPN) consistently decreased (by -46, -48 and -41% respectively) the Pitx1 mRNA in the anterior pituitary gland. Orchestrated Egr1 and Pitx1 activities depending on specific central and peripheral regulatory inputs could be responsible for physiologically variable Lhb gene promoter activation in vivo. © 2014 Society for Endocrinology.

  18. Using Dual Fluorescence Reporting Genes to Establish an In Vivo Imaging Model of Orthotopic Lung Adenocarcinoma in Mice.

    Science.gov (United States)

    Lai, Cheng-Wei; Chen, Hsiao-Ling; Yen, Chih-Ching; Wang, Jiun-Long; Yang, Shang-Hsun; Chen, Chuan-Mu

    2016-12-01

    Lung adenocarcinoma is characterized by a poor prognosis and high mortality worldwide. In this study, we purposed to use the live imaging techniques and a reporter gene that generates highly penetrative near-infrared (NIR) fluorescence to establish a preclinical animal model that allows in vivo monitoring of lung cancer development and provides a non-invasive tool for the research on lung cancer pathogenesis and therapeutic efficacy. A human lung adenocarcinoma cell line (A549), which stably expressed the dual fluorescence reporting gene (pCAG-iRFP-2A-Venus), was used to generate subcutaneous or orthotopic lung cancer in nude mice. Cancer development was evaluated by live imaging via the NIR fluorescent signals from iRFP, and the signals were verified ex vivo by the green fluorescence of Venus from the gross lung. The tumor-bearing mice received miR-16 nucleic acid therapy by intranasal administration to demonstrate therapeutic efficacy in this live imaging system. For the subcutaneous xenografts, the detection of iRFP fluorescent signals revealed delicate changes occurring during tumor growth that are not distinguishable by conventional methods of tumor measurement. For the orthotopic xenografts, the positive correlation between the in vivo iRFP signal from mice chests and the ex vivo green fluorescent signal from gross lung tumors and the results of the suppressed tumorigenesis by miR-16 treatment indicated that lung tumor size can be accurately quantified by the emission of NIR fluorescence. In addition, orthotopic lung tumor localization can be accurately visualized using iRFP fluorescence tomography in vivo, thus revealing the trafficking of lung tumor cells. We introduced a novel dual fluorescence lung cancer model that provides a non-invasive option for preclinical research via the use of NIR fluorescence in live imaging of lung.

  19. In Vivo Imaging of Retinoic Acid Receptor Activity using a Sodium/Iodide Symporter and Luciferase Dual Imaging Reporter Gene

    Directory of Open Access Journals (Sweden)

    Min Kyung So

    2004-07-01

    Full Text Available Retinoic acids are natural derivatives of vitamin A, and play important roles in modulating tumor cell growth by regulating differentiation, thus suggesting the potential use of these derivatives in cancer therapy and prevention. To visualize the intranuclear responses of functional retinoic acid receptors, we have developed a dual-imaging reporter gene system based on the use of sodium/iodide symporter (NIS and luciferase in cancer cell lines. NIS and luciferase genes were linked with an internal ribosome entry site, and placed under the control of an artificial cis-acting retinoic acid responsive element (pRARE/NL. After retinoic acid treatment, I-125 uptake by pRARE/NL transfected cells was found to have increased by up to about five times that of nontreated cells. The bioluminescence intensity of pRARE/NL transfected cells showed dose-dependency. In vivo luciferase images showed higher intensity in retinoic acid treated SK-RARE/NL tumors, and scintigraphic images of SK-RARE/NL tumors showed increased Tc-99m uptake after retinoic acid treatment. The NIS/luciferase imaging reporter system was sufficiently sensitive to allow the visualization of intranuclear retinoic acid receptor activity. This cis-enhancer imaging reporter system may be useful in vitro and in vivo for the evaluation of retinoic acid responses in such areas as cellular differentiation and chemoprevention.

  20. Effective in vitro and in vivo gene delivery by the combination of liposomal bubbles (bubble liposomes) and ultrasound exposure.

    Science.gov (United States)

    Suzuki, Ryo; Maruyama, Kazuo

    2010-01-01

    Gene delivery with a physical mechanism using ultrasound (US) and nano/microbubbles is expected as an ideal system in terms of delivering plasmid DNA noninvasively into a specific target site. We developed novel liposomal bubbles (Bubble liposomes (BLs)) containing the lipid nanobubbles of perfluoropropane which were utilized for contrast enhancement in ultrasonography. BLs were smaller in diameter than conventional microbubbles and induced cavitation upon exposure ultrasound. In addition, when coupled with US exposure, BLs could deliver plasmid DNA into various types of cells in vitro and in vivo. The transfection efficiency with BLs and US was higher than that with conventional lipofection method. Therefore, the combination of BLs and US might be an efficient and novel nonviral gene delivery system.

  1. A Histone-Like Protein Induces Plasmid DNA to Form Liquid Crystals in Vitro and Gene Compaction in Vivo

    Directory of Open Access Journals (Sweden)

    Shiyong Sun

    2013-12-01

    Full Text Available The liquid crystalline state is a universal phenomenon involving the formation of an ordered structure via a self-assembly process that has attracted attention from numerous scientists. In this study, the dinoflagellate histone-like protein HCcp3 is shown to induce super-coiled pUC18 plasmid DNA to enter a liquid crystalline state in vitro, and the role of HCcp3 in gene condensation in vivo is also presented. The plasmid DNA (pDNA-HCcp3 complex formed birefringent spherical particles with a semi-crystalline selected area electronic diffraction (SAED pattern. Circular dichroism (CD titrations of pDNA and HCcp3 were performed. Without HCcp3, pUC18 showed the characteristic B conformation. As the HCcp3 concentration increased, the 273 nm band sharply shifted to 282 nm. When the HCcp3 concentration became high, the base pair (bp/dimer ratio fell below 42/1, and the CD spectra of the pDNA-HCcp3 complexes became similar to that of dehydrated A-form DNA. Microscopy results showed that HCcp3 compacted the super-coiled gene into a condensed state and that inclusion bodies were formed. Our results indicated that HCcp3 has significant roles in gene condensation both in vitro and in histone-less eukaryotes in vivo. The present study indicates that HCcp3 has great potential for applications in non-viral gene delivery systems, where HCcp3 may compact genetic material to form liquid crystals.

  2. Nonviral gene therapy in vivo with PAM-RG4/apoptin as a potential brain tumor therapeutic.

    Science.gov (United States)

    An, Songhie; Nam, Kihoon; Choi, Sunghyun; Bai, Cheng Z; Lee, Yan; Park, Jong-Sang

    2013-01-01

    Glioma is still one of the most complicated forms of brain tumor to remove completely due to its location and the lack of an efficient means to specifically eliminate tumor cells. For these reasons, this study has examined the effectiveness of a nonviral gene therapy approach utilizing a tumor-selective killer gene on a brain tumor xenograft model. The therapeutic apoptin gene was recombined into the JDK plasmid and delivered into human brain tumor cells (U87MG) by using a polyamidoamine dendrimer with an arginine surface (PAM-RG4). Studies in vitro showed that the PAM-RG4/apoptin plasmid polyplex exhibited a particularly high transfection activity of .40%. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, 4',6-Diamidino-2-phenylindole (DAPI) TUNEL assay, DAPI staining, and caspase-3 activity assay verified that the tumor cells had undergone apoptosis induced by apoptin. For in vivo studies, the polyplex was injected into tumors, which were induced by injecting U87MG cells intradermally into nude mice. Based on hematoxylin and eosin staining, epidermal growth factor receptor immunohistochemistry results and tumor volume measurement results, tumor growth was effectively inhibited and no specific edema, irritation, or other harm to the skin was observed after polyplex injection. The in vivo expression of apoptin and the induction of apoptosis were verified by reverse-transcription polymerase chain reaction analysis, TUNEL assay, and DAPI staining. The PAM-RG4/apoptin gene polyplex is a strong candidate for brain tumor therapeutics because of the synergistic effect of the carrier's high transfection efficiency (35%-40%) in glioma cells and the selective apoptosis-inducing activity of apoptin in tumor cells.

  3. Effects of Doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo

    Directory of Open Access Journals (Sweden)

    Rao Ramakrishna U

    2012-02-01

    Full Text Available Abstract Background Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi. Methods Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles. Results and discussion Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%. In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes. Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion. Conclusions Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron

  4. In vivo image of radioiodinated IVDU and IVFRU in HSV-TK gene tranduced hepatocellular carcinoma bearing buffalo rat

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tae Sup; Choi, T. H.; Ahn, S. H.; Woo, K. S.; Chung, W. S.; Lee, S. J.; Choi, C. W. [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    2000-07-01

    The extent of gene delivery and expression in gene therapy with suicide genes such as herpes simplex virus thymidine kinase (HSV-tk) is assessed with measurement of selective localization of radioiodinated HSV-tk substrates in HSV-tk expressing tumor. We compared n vitro uptake of {sup 125}I-IVDU, IVFRU and in vivo image of HSV-tk gene tranduced hepatocellular carcinoma model. Using H{sub 2}O{sub 2}(hydrogen peroxide), IVDU and IVFRU was radiolabeled as carrier free form. The uptake of {sup 125}I-IVDU IVFRU was determined with increasing incubation periods in MCA-tk and MCA cell line (1X10{sup 6}cell/flask). The cell harvested and counted after incubation of 15, 30, 60, 120, 240, 480 minutes. For estimating accumulation of radiolabelled IVDU, IVFRU in HSV-tk expressing tumor, MCA-tk cells (1 X 10{sup 6}/100 {mu}l) injected intramuscularly into right thigh of buffalo rats. To determine selective localization of radiolabelled IVDU, IVFRU in HSV-tk expressing hepatocellular carcinoma bearing buffalo rats, MCA-tk cells (1X 10{sup 7} cell/100 {mu}l) were injected subcutaneously into both shoulders of buffalo rats. Established tumor mass implanted into liver of buffalo rats using intra-hepatic tumor injection. Two weeks later, {sup 123}I labelled IVDU, IVFRU(7.4 X 10{sup 7}Bq/200 {mu}l) injected intravenously into tail veins of each buffalo rats. Gamma camera used as revealing localization of {sup 123}I-IVDU, IVFRU in MCA-tk cells grafts rats and in vivo image was taken 2 hrs, 24 hrs after injection. radioiodinated IVDU, IVFRU were radiolabeled with {sup 123}I as labeling yield 70%, {sup 125}I as 84%. Two compounds showed minimal uptake in MCA cell line, but in MCA-tk cell line, increased uptake was observed. The ratio of MCA-tk to MCA was up to 116-fold in {sup 125}I-IVDU, up to 37-fold in {sup 125}I-IVFRU at 480 min. The uptake of IVDU was 4 times higher than IVFRU in MCA-tk cells. Gamma camera images of HSV-tk gene tranduced MCA tumor showed accumulation of {sup 123}I

  5. Candidate gene identification of ovulation-inducing genes by RNA sequencing with an in vivo assay in zebrafish.

    Directory of Open Access Journals (Sweden)

    Wanlada Klangnurak

    Full Text Available We previously reported the microarray-based selection of three ovulation-related genes in zebrafish. We used a different selection method in this study, RNA sequencing analysis. An additional eight up-regulated candidates were found as specifically up-regulated genes in ovulation-induced samples. Changes in gene expression were confirmed by qPCR analysis. Furthermore, up-regulation prior to ovulation during natural spawning was verified in samples from natural pairing. Gene knock-out zebrafish strains of one of the candidates, the starmaker gene (stm, were established by CRISPR genome editing techniques. Unexpectedly, homozygous mutants were fertile and could spawn eggs. However, a high percentage of unfertilized eggs and abnormal embryos were produced from these homozygous females. The results suggest that the stm gene is necessary for fertilization. In this study, we selected additional ovulation-inducing candidate genes, and a novel function of the stm gene was investigated.

  6. Pregnancy-induced gene expression changes in vivo among women with rheumatoid arthritis: a pilot study.

    Science.gov (United States)

    Goin, Dana E; Smed, Mette Kiel; Pachter, Lior; Purdom, Elizabeth; Nelson, J Lee; Kjærgaard, Hanne; Olsen, Jørn; Hetland, Merete Lund; Zoffmann, Vibeke; Ottesen, Bent; Jawaheer, Damini

    2017-05-25

    Little is known about gene expression changes induced by pregnancy in women with rheumatoid arthritis (RA) and healthy women because the few studies previously conducted did not have pre-pregnancy samples available as baseline. We have established a cohort of women with RA and healthy women followed prospectively from a pre-pregnancy baseline. In this study, we tested the hypothesis that pregnancy-induced changes in gene expression among women with RA who improve during pregnancy (pregDAS improved ) overlap substantially with changes observed among healthy women and differ from changes observed among women with RA who worsen during pregnancy (pregDAS worse ). Global gene expression profiles were generated by RNA sequencing (RNA-seq) from 11 women with RA and 5 healthy women before pregnancy (T0) and at the third trimester (T3). Among the women with RA, eight showed an improvement in disease activity by T3, whereas three worsened. Differential expression analysis was used to identify genes demonstrating significant changes in expression within each of the RA and healthy groups (T3 vs T0), as well as between the groups at each time point. Gene set enrichment was assessed in terms of Gene Ontology processes and protein networks. A total of 1296 genes were differentially expressed between T3 and T0 among the 8 pregDAS improved women, with 161 genes showing at least two-fold change (FC) in expression by T3. The majority (108 of 161 genes) were also differentially expressed among healthy women (qexpression between the pregDAS improved and pregDAS worse groups, all of which were inducible by type I interferon (IFN). These IFN-inducible genes were over-expressed at T3 compared to the T0 baseline among the pregDAS improved women. In our pilot RNA-seq dataset, increased pregnancy-induced expression of type I IFN-inducible genes was observed among women with RA who improved during pregnancy, but not among women who worsened. These findings warrant further investigation into

  7. [Identification and detection of trag: a new infection-related gene expressed in vivo from isolates of Streptococcus suis].

    Science.gov (United States)

    Zhu, Haodan; Gu, Hongwei; Lu, Chengping

    2008-12-01

    The trag (transfer gene G) was one of the novel infection-related factors identified by in vivo-induced antigen technology (IVIAT) from Streptococcus suis type 2 expression libraries with swine convalesecent sera in our former research. We detected the distribution of trag in different Streptococcus suis isolates and identify the differential expression of the new infection-related factor between in vivo and in vitro condition. According to the sequence of trag of North American strain 89/1591, a pair of primers were designed to detect the distribution of trag in total 43 SS isolates. Another pair of primers were designed to amplify the ORF of trag of 5 SS representive strains (ZY05719, HA9801, 98012, SH040805, SH040917). Partial gene of trag was cloned and inserted into expression vector pET28a(+), and induced by IPTG to express recombinant TRAG. The recombinant protein was probed with swine convalescent sera and immune sera respectively. The trag was detected in the most of SS2 isolates (30/32), in SS9 isolates (4/6), and 1 isolate of SS7, while it was not found in SS2 European strain ATCC43765, avirulent strain SS2 T15, 1 isolates of SS1, 1 isolates of SS1/2 and 2 isolates of group C streptococcal strains from pigs. Comparisons between the sequences of TRAG of 5 isolates with that of SS isolates, showed a high homology (>97%) with North American strain 89/1589 and China strains 98HAH33, 05ZYH33. The immunoreactivity was only presented with convalescent sera. The trag was detected from virulent SS isolates but not from avirulent strain, which suggested that this gene may be related to the pathogenicity of SS. The special reactivity was only present with convalescent sera, and it indicated that TRAG might play a role during SS2 invasive course.

  8. In vivo and in vitro experimental study on cervix cancer with combination of HSV-TK/GCV suicide gene therapy system and 60Co radiotherapy

    International Nuclear Information System (INIS)

    Chen Daozhen; Xue Wenqun; Zhan Huiying; Zhu Yunxia; Yang Youyi; Liu Lu; Tang Qiusha

    2006-01-01

    Objective: To evaluate the killing effect of HSV-TK/GCV suicide gene therapy system combined with 60 Co radiotherapy on human cervical cancer HeLa cell line in vivo and in vitro, and to explore radiosensitization by the HSV-TK/GCV system. Methods: The HSV-TK/GCV suicide gene therapy system and 60 Co radiotherapy were used separately or in combination for human cervical cancer HeLa cell line in vivo and in vitro to compare their effects. Colony formation test and the rate of radiosensitization effect(E/O) were employed to observed the radiosensitization by the HSV-TK/GCV system. Results: The HSV-TK/GCV suicide gene therapy system showed strong therapeutic effects on HeLa cells both in vitro and in vivo (the inhibition rates were 45.8% and 39.5%, respectively). Moreover, the combined application of gene therapy and radiotherapy exhibited stronger therapeutic effects in vitro and in vivo (the inhibition rate was 87.5% in vitro, and was 87.9% in vivo) (P 1.4), indicating the HSV-TK/GCV system could exert a sensitizing effect on 60 Co radiotherapy of the transplanted human cervical cancer cells in nude mice. Conclusion: The HSV-TK/GCV system has radiosensitizationaction. Gene therapy combined with radiotherapy may be a good supplementary method for synthetic treatment of cervical cancer. (authors)

  9. Nanostructured implant surface effect on osteoblast gene expression and bone-to-implant contact in vivo

    International Nuclear Information System (INIS)

    Mendonca, Gustavo; Baccelli Silveira Mendonca, Daniela; Pagotto Simoes, Luis Gustavo; Araujo, Andre Luis; Leite, Edson Roberto; Golin, Alexsander Luiz; Aragao, Francisco J.L.; Cooper, Lyndon F.

    2011-01-01

    The aim of this study was to investigate the response of nanostructured implant surfaces at the level of osteoblast differentiation and its effects in bone-to-implant contact (BIC) and removal-torque values (RTV). CpTi grade IV implants (1.6 x 4.0 mm) were machined or machined and subsequently coated with an oxide solution. The surfaces were divided into: machined (M), titania-anatase (An), titania-rutile (Ru), and zirconia (Zr). Surfaces were examined by scanning electron microscopy, atomic force microscopy, and by X-ray microanalysis. Implants were inserted in rat tibia and harvested from 0 to 21 days for measurement of Alkaline Phosphatase, Bone Sialoprotein, Osteocalcin, Osteopontin, and RUNX-2 mRNA levels by real time PCR; from 0 to 56 days for RTV; and from 0 to 56 days for BIC. The roughness parameter (Sa) was compared by one-way ANOVA followed by Tukey Test. Comparison of Torque removal values and histomorphometric measurements on implants in vivo was performed by Kruskal-Wallis test and the significance level for all statistical analyses was set at p ≤ 0.05. mRNA levels on all nanostructured surfaces were increased compared to M. At 56 days, the mean RTV in Ncm was 11.6 ± 2.5, 11.3 ± 2.4, 11.1 ± 3.5, 9.7 ± 1.4 for An, Ru, Zr, and M, respectively. Higher BIC (%) was measured for all the nanostructured surfaces versus M at 21 and 56 days (p 2 or ZrO 2 applied to machined cpTi implant promoted greater mesenchymal stem cell commitment to the osteoblast phenotype and associated increased BIC and physical association with bone. Highlights: → Nanostructured surfaces using a sol-gel technique coated cpTi with TiO 2 or ZrO 2 . → Evaluated molecular and mechanical effect of nanofeatures in vivo in rat tibiae. → Nanofeatures improved the differentiation of rat MSCs into osteoblasts. → Nanofeatures improved increased bone-to-implant contact and removal torque values. → TiO 2 or ZrO 2 nanofeatures improved the biological response of machined titanium.

  10. Nanostructured implant surface effect on osteoblast gene expression and bone-to-implant contact in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Mendonca, Gustavo, E-mail: gustavo_mendonca@dentistry.unc.edu [Universidade Catolica de Brasilia, Pos-Graduacao em Ciencias Genomicas e Biotecnologia, SGAN Quadra 916, Modulo B, Av. W5 Norte 70.790-160-Asa Norte Brasilia/DF (Brazil); Bone Biology and Implant Therapy Laboratory, Department of Prosthodontics, University of North Carolina at Chapel Hill, 404 Brauer Hall, CB 7450, Chapel Hill, NC 27511 (United States); Universidade Catolica de Brasilia, Curso de Odontologia, Taguatinga/DF (Brazil); Baccelli Silveira Mendonca, Daniela [Universidade Catolica de Brasilia, Pos-Graduacao em Ciencias Genomicas e Biotecnologia, SGAN Quadra 916, Modulo B, Av. W5 Norte 70.790-160-Asa Norte Brasilia/DF (Brazil) and Bone Biology and Implant Therapy Laboratory, Department of Prosthodontics, University of North Carolina at Chapel Hill, 404 Brauer Hall, CB 7450, Chapel Hill, NC 27511 (United States); Pagotto Simoes, Luis Gustavo; Araujo, Andre Luis; Leite, Edson Roberto [Departmento de Quimica, Universidade Federal de Sao Carlos-UFSCAR, Rod. Washington Luiz, 13565-905 Sao Carlos, SP (Brazil); Golin, Alexsander Luiz [Departmento de Engenharia Mecanica, Faculdade de Engenharia Mecanica, Pontificia Universidade Catolica de Curitiba, Curitiba, PR (Brazil); Aragao, Francisco J.L. [Universidade Catolica de Brasilia, Pos-Graduacao em Ciencias Genomicas e Biotecnologia, SGAN Quadra 916, Modulo B, Av. W5 Norte 70.790-160-Asa Norte Brasilia/DF (Brazil); Embrapa Recursos Geneticos e Biotecnologia, Laboratorio de Introducao e Expressao de Genes, PqEB W5 Norte, 70770-900, Brasilia, DF (Brazil); Cooper, Lyndon F., E-mail: lyndon_cooper@dentistry.unc.edu [Bone Biology and Implant Therapy Laboratory, Department of Prosthodontics, University of North Carolina at Chapel Hill, 404 Brauer Hall, CB 7450, Chapel Hill, NC 27511 (United States)

    2011-12-01

    The aim of this study was to investigate the response of nanostructured implant surfaces at the level of osteoblast differentiation and its effects in bone-to-implant contact (BIC) and removal-torque values (RTV). CpTi grade IV implants (1.6 x 4.0 mm) were machined or machined and subsequently coated with an oxide solution. The surfaces were divided into: machined (M), titania-anatase (An), titania-rutile (Ru), and zirconia (Zr). Surfaces were examined by scanning electron microscopy, atomic force microscopy, and by X-ray microanalysis. Implants were inserted in rat tibia and harvested from 0 to 21 days for measurement of Alkaline Phosphatase, Bone Sialoprotein, Osteocalcin, Osteopontin, and RUNX-2 mRNA levels by real time PCR; from 0 to 56 days for RTV; and from 0 to 56 days for BIC. The roughness parameter (Sa) was compared by one-way ANOVA followed by Tukey Test. Comparison of Torque removal values and histomorphometric measurements on implants in vivo was performed by Kruskal-Wallis test and the significance level for all statistical analyses was set at p {<=} 0.05. mRNA levels on all nanostructured surfaces were increased compared to M. At 56 days, the mean RTV in Ncm was 11.6 {+-} 2.5, 11.3 {+-} 2.4, 11.1 {+-} 3.5, 9.7 {+-} 1.4 for An, Ru, Zr, and M, respectively. Higher BIC (%) was measured for all the nanostructured surfaces versus M at 21 and 56 days (p < 0.05). Nanostructured topographic features composed of TiO{sub 2} or ZrO{sub 2} applied to machined cpTi implant promoted greater mesenchymal stem cell commitment to the osteoblast phenotype and associated increased BIC and physical association with bone. Highlights: {yields} Nanostructured surfaces using a sol-gel technique coated cpTi with TiO{sub 2} or ZrO{sub 2}. {yields} Evaluated molecular and mechanical effect of nanofeatures in vivo in rat tibiae. {yields} Nanofeatures improved the differentiation of rat MSCs into osteoblasts. {yields} Nanofeatures improved increased bone-to-implant contact and

  11. siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma.

    Science.gov (United States)

    Ramon, A L; Bertrand, J R; de Martimprey, H; Bernard, G; Ponchel, G; Malvy, C; Vauthier, C

    2013-07-01

    Ewing's sarcoma is a rare, mostly pediatric bone cancer that presents a chromosome abnormality called EWS/Fli-1, responsible for the development of the tumor. In vivo, tumor growth can be inhibited specifically by delivering small interfering RNA (siRNA) associated with nanoparticles. The aim of the work was to design targeted nanoparticles against the cell membrane glycoprotein cd99, which is overexpressed in Ewing's sarcoma cells to improve siRNA delivery to tumor cells. Biotinylated poly(isobutylcyanoacrylate) nanoparticles were conceived as a platform to design targeted nanoparticles with biotinylated ligands and using the biotin-streptavidin coupling method. The targeted nanoparticles were validated in vivo for the targeted delivery of siRNA after systemic administration to mice bearing a tumor model of the Ewing's sarcoma. The expression of the gene responsible of Ewing's sarcoma was inhibited at 78% ± 6% by associating the siRNA with the cd99-targeted nanoparticles compared with an inhibition of only 41% ± 9% achieved with the nontargeted nanoparticles. Copyright © 2013 John Wiley & Sons, Ltd.

  12. Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus.

    Science.gov (United States)

    Flannery, J G; Zolotukhin, S; Vaquero, M I; LaVail, M M; Muzyczka, N; Hauswirth, W W

    1997-06-24

    We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10-20% of the total retinal area after a single 2-microl injection. Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The gfp-containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.

  13. Circadian Enhancers Coordinate Multiple Phases of Rhythmic Gene Transcription In Vivo

    Science.gov (United States)

    Fang, Bin; Everett, Logan J.; Jager, Jennifer; Briggs, Erika; Armour, Sean M.; Feng, Dan; Roy, Ankur; Gerhart-Hines, Zachary; Sun, Zheng; Lazar, Mitchell A.

    2014-01-01

    SUMMARY Mammalian transcriptomes display complex circadian rhythms with multiple phases of gene expression that cannot be accounted for by current models of the molecular clock. We have determined the underlying mechanisms by measuring nascent RNA transcription around the clock in mouse liver. Unbiased examination of eRNAs that cluster in specific circadian phases identified functional enhancers driven by distinct transcription factors (TFs). We further identify on a global scale the components of the TF cistromes that function to orchestrate circadian gene expression. Integrated genomic analyses also revealed novel mechanisms by which a single circadian factor controls opposing transcriptional phases. These findings shed new light on the diversity and specificity of TF function in the generation of multiple phases of circadian gene transcription in a mammalian organ. PMID:25416951

  14. Nonviral gene therapy in vivo with PAM-RG4/apoptin as a potential brain tumor therapeutic

    Directory of Open Access Journals (Sweden)

    An S

    2013-02-01

    Full Text Available Songhie An,* Kihoon Nam,* Sunghyun Choi, Cheng Z Bai, Yan Lee, Jong-Sang ParkDepartment of Chemistry, Seoul National University, Seoul, Republic of Korea*These authors contributed equally to this workBackground: Glioma is still one of the most complicated forms of brain tumor to remove completely due to its location and the lack of an efficient means to specifically eliminate tumor cells. For these reasons, this study has examined the effectiveness of a nonviral gene therapy approach utilizing a tumor-selective killer gene on a brain tumor xenograft model.Methods and results: The therapeutic apoptin gene was recombined into the JDK plasmid and delivered into human brain tumor cells (U87MG by using a polyamidoamine dendrimer with an arginine surface (PAM-RG4. Studies in vitro showed that the PAM-RG4/apoptin plasmid polyplex exhibited a particularly high transfection activity of >40%. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay, 4´,6-Diamidino-2-phenylindole (DAPI TUNEL assay, DAPI staining, and caspase-3 activity assay verified that the tumor cells had undergone apoptosis induced by apoptin. For in vivo studies, the polyplex was injected into tumors, which were induced by injecting U87MG cells intradermally into nude mice. Based on hematoxylin and eosin staining, epidermal growth factor receptor immunohistochemistry results and tumor volume measurement results, tumor growth was effectively inhibited and no specific edema, irritation, or other harm to the skin was observed after polyplex injection. The in vivo expression of apoptin and the induction of apoptosis were verified by reverse-transcription polymerase chain reaction analysis, TUNEL assay, and DAPI staining.Conclusion: The PAM-RG4/apoptin gene polyplex is a strong candidate for brain tumor therapeutics because of the synergistic effect of the carrier's high transfection efficiency (35%–40% in glioma cells and the selective apoptosis-inducing activity of

  15. Pregnancy-induced gene expression changes in vivo among women with rheumatoid arthritis

    DEFF Research Database (Denmark)

    Goin, Dana E; Smed, Mette Kiel; Pachter, Lior

    2017-01-01

    BACKGROUND: Little is known about gene expression changes induced by pregnancy in women with rheumatoid arthritis (RA) and healthy women because the few studies previously conducted did not have pre-pregnancy samples available as baseline. We have established a cohort of women with RA and healthy...

  16. GATA-2 and GATA-3 regulate trophoblast-specific gene expression in vivo.

    NARCIS (Netherlands)

    G.T. Ma (Grace); M.E. Roth (Matthew); J.C. Groskopf (John); F.G. Grosveld (Frank); J.D. Engel (Douglas); D.I.H. Linzer (Daniel); F.Y. Tsai (Fong-Ying); S.H. Orkin (Stuart)

    1997-01-01

    textabstractWe previously demonstrated that the zinc finger transcription factors GATA-2 and GATA-3 are expressed in trophoblast giant cells and that they regulate transcription from the mouse placental lactogen I gene promoter in a transfected trophoblast cell line. We present evidence here that

  17. Triple-Gene Therapy for Stroke: A Proof-of-Concept in Vivo Study in Rats

    Directory of Open Access Journals (Sweden)

    Mikhail E. Sokolov

    2018-02-01

    Full Text Available Natural brain repair after stroke is extremely limited, and current therapeutic options are even more scarce with no clinical break-through in sight. Despite restricted regeneration in the central nervous system, we have previously proved that human umbilical cord blood mono-nuclear cells (UCB-MC transduced with adenoviral vectors carrying genes encoding vascular endothelial growth factor (VEGF, glial cell-derived neurotrophic factor (GDNF, and neural cell adhesion molecule (NCAM successfully rescued neurons in amyotrophic lateral sclerosis and spinal cord injury. This proof-of-principle project was aimed at evaluating the beneficial effects of the same triple-gene approach in stroke. Rats subjected to distal occlusion of the middle cerebral artery were treated intrathecally with a combination of these genes either directly or using our cell-based (UCB-MC approach. Various techniques and markers were employed to evaluate brain injury and subsequent recovery after treatment. Brain repair was most prominent when therapeutic genes were delivered via adenoviral vector- or UCB-MC-mediated approach. Remodeling of brain cortex in the stroke area was confirmed by reduction of infarct volume and attenuated neural cell death, depletion of astrocytes and microglial cells, and increase in the number of oligodendroglial cells and synaptic proteins expression. These results imply that intrathecal injection of genetically engineered UCB-MC over-expressing therapeutic molecules (VEGF, GDNF, and NCAM following cerebral blood vessel occlusion might represent a novel avenue for future research into treating stroke.

  18. Expression analysis of genes implicated in meiotic resumption in vivo and developmental competence

    NARCIS (Netherlands)

    Algriany, O.A.

    2007-01-01

    This thesis investigated the gene expression in bovine oocytes during meiotic resumption, at 6 h post LH surge, coinciding with germinal vesicle breakdown, which was supposed to give a picture of the major cell cycle regulation changes, cytoskeleton rearrangement and chromosome alignment.

  19. Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia

    Directory of Open Access Journals (Sweden)

    Serena Joaquín

    2009-06-01

    Full Text Available Abstract Background Studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. The normalization of relative qRT-PCR data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used. The present study aims to determine the expression stability of ten housekeeping genes (Gapdh, β2m, Hprt, Ppia, Rpl13a, Oaz1, 18S rRNA, Gusb, Ywhaz and Sdha to establish their suitability as control genes for in-vitro and in-vivo cerebral ischaemia models. Results The expression stability of the candidate reference genes was evaluated using the 2-ΔC'T method and ANOVA followed by Dunnett's test. For the in-vitro model using primary cultures of rat astrocytes, all genes analysed except for Rpl13a and Sdha were found to have significantly different levels of mRNA expression. These different levels were also found in the case of the in-vivo model of pMCAO in rats except for Hprt, Sdha and Ywhaz mRNA, where the expression did not vary. Sdha and Ywhaz were identified by geNorm and NormFinder as the two most stable genes. Conclusion We have validated endogenous control genes for qRT-PCR analysis of gene expression in in-vitro and in-vivo cerebral ischaemia models. For normalization purposes, Rpl13a and Sdha are found to be the most suitable genes for the in-vitro model and Sdha and Ywhaz for the in-vivo model. Genes previously used as housekeeping genes for the in-vivo model in the literature were not validated as good control genes in the present study, showing the need for careful evaluation for each new experimental setup.

  20. Human cancer cells express Slug-based epithelial-mesenchymal transition gene expression signature obtained in vivo

    International Nuclear Information System (INIS)

    Anastassiou, Dimitris; Rumjantseva, Viktoria; Cheng, Weiyi; Huang, Jianzhong; Canoll, Peter D; Yamashiro, Darrell J; Kandel, Jessica J

    2011-01-01

    The biological mechanisms underlying cancer cell motility and invasiveness remain unclear, although it has been hypothesized that they involve some type of epithelial-mesenchymal transition (EMT). We used xenograft models of human cancer cells in immunocompromised mice, profiling the harvested tumors separately with species-specific probes and computationally analyzing the results. Here we show that human cancer cells express in vivo a precise multi-cancer invasion-associated gene expression signature that prominently includes many EMT markers, among them the transcription factor Slug, fibronectin, and α-SMA. We found that human, but not mouse, cells express the signature and Slug is the only upregulated EMT-inducing transcription factor. The signature is also present in samples from many publicly available cancer gene expression datasets, suggesting that it is produced by the cancer cells themselves in multiple cancer types, including nonepithelial cancers such as neuroblastoma. Furthermore, we found that the presence of the signature in human xenografted cells was associated with a downregulation of adipocyte markers in the mouse tissue adjacent to the invasive tumor, suggesting that the signature is triggered by contextual microenvironmental interactions when the cancer cells encounter adipocytes, as previously reported. The known, precise and consistent gene composition of this cancer mesenchymal transition signature, particularly when combined with simultaneous analysis of the adjacent microenvironment, provides unique opportunities for shedding light on the underlying mechanisms of cancer invasiveness as well as identifying potential diagnostic markers and targets for metastasis-inhibiting therapeutics

  1. Effective in vivo and ex vivo gene transfer to intestinal mucosa by VSV-G-pseudotyped lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Kasahara Noriyuki

    2010-05-01

    Full Text Available Abstract Background Gene transfer to the gastrointestinal (GI mucosa is a therapeutic strategy which could prove particularly advantageous for treatment of various hereditary and acquired intestinal disorders, including inflammatory bowel disease (IBD, GI infections, and cancer. Methods We evaluated vesicular stomatitis virus glycoprotein envelope (VSV-G-pseudotyped lentiviral vectors (LV for efficacy of gene transfer to both murine rectosigmoid colon in vivo and human colon explants ex vivo. LV encoding beta-galactosidase (LV-β-Gal or firefly-luciferase (LV-fLuc reporter genes were administered by intrarectal instillation in mice, or applied topically for ex vivo transduction of human colorectal explant tissues from normal individuals. Macroscopic and histological evaluations were performed to assess any tissue damage or inflammation. Transduction efficiency and systemic biodistribution were evaluated by real-time quantitative PCR. LV-fLuc expression was evaluated by ex vivo bioluminescence imaging. LV-β-Gal expression and identity of transduced cell types were examined by histochemical and immunofluorescence staining. Results Imaging studies showed positive fLuc signals in murine distal colon; β-Gal-positive cells were found in both murine and human intestinal tissue. In the murine model, β-Gal-positive epithelial and lamina propria cells were found to express cytokeratin, CD45, and CD4. LV-transduced β-Gal-positive cells were also seen in human colorectal explants, consisting mainly of CD45, CD4, and CD11c-positive cells confined to the LP. Conclusions We have demonstrated the feasibility of LV-mediated gene transfer into colonic mucosa. We also identified differential patterns of mucosal gene transfer dependent on whether murine or human tissue was used. Within the limitations of the study, the LV did not appear to induce mucosal damage and were not distributed beyond the distal colon.

  2. In vivo functional requirement of the mouse Ifitm1 gene for germ cell development, interferon mediated immune response and somitogenesis.

    Directory of Open Access Journals (Sweden)

    Ingeborg Klymiuk

    Full Text Available The mammalian Interferon induced transmembrane protein 1 (Ifitm1 gene was originally identified as a member of a gene family highly inducible by type I and type II interferons. Based on expression analyses, it was suggested to be required for normal primordial germ cell migration. The knockdown of Ifitm1 in mouse embryos provided evidence for a role in somitogenesis. We generated the first targeted knockin allele of the Ifitm1 gene to systematically reassess all inferred functions. Sperm motility and the fertility of male and female mutant mice are as in wild type littermates. Embryonic somites and the adult vertebral column appear normal in homozygous Ifitm1 knockout mice, demonstrating that Ifitm1 is not essential for normal segmentation of the paraxial mesoderm. Proportions of leucocyte subsets, including granulocytes, monocytes, B-cells, T-cells, NK-cells, and NKT-cells, are unchanged in mutant mice. Based on a normal immune response to Listeria monocytogenes infection, there is no evidence for a dysfunction in downstream IFNγ signaling in Ifitm1 mutant mice. Expression from the Ifitm1 locus from E8.5 to E14.5 is highly dynamic. In contrast, in adult mice, Ifitm1 expression is highly restricted and strong in the bronchial epithelium. Intriguingly, IFITM1 is highly overexpressed in tumor epithelia cells of human squamous cell carcinomas and in adenocarcinomas of NSCLC patients. These analyses underline the general importance of targeted in vivo studies for the functional annotation of the mammalian genome. The first comprehensive description of the Ifitm1 expression pattern provides a rational basis for the further examination of Ifitm1 gene functions. Based on our data, the fact that IFITM1 can function as a negative regulator of cell proliferation, and because the gene maps to chromosome band 11p15.5, previously associated with NSCLC, it is likely that IFITM1 in man has a key role in tumor formation.

  3. Hypoxia targeted bifunctional suicide gene expression enhances radiotherapy in vitro and in vivo

    International Nuclear Information System (INIS)

    Sun, Xiaorong; Xing, Ligang; Deng, Xuelong; Hsiao, Hung Tsung; Manami, Akiko; Koutcher, Jason A.; Clifton Ling, C.; Li, Gloria C.

    2012-01-01

    Purpose: To investigate whether hypoxia targeted bifunctional suicide gene expression-cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) with 5-FC treatments can enhance radiotherapy. Materials and methods: Stable transfectants of R3327-AT cells were established which express a triple-fusion-gene: CD, UPRT and monomoric DsRed (mDsRed) controlled by a hypoxia inducible promoter. Hypoxia-induced expression/function of CDUPRTmDsRed was verified by western blot, flow cytometry, fluorescent microscopy, and cytotoxicity assay of 5-FU and 5-FC. Tumor-bearing mice were treated with 5-FC and local radiation. Tumor volume was monitored and compared with those treated with 5-FC or radiation alone. In addition, the CDUPRTmDsRed distribution in hypoxic regions of tumor sections was visualized with fluorescent microscopy. Results: Hypoxic induction of CDUPRTmDsRed protein correlated with increased sensitivity to 5-FC and 5-FU. Significant radiosensitization effects were detected after 5-FC treatments under hypoxic conditions. In the tumor xenografts, the distribution of CDUPRTmDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC to mice in combination with local irradiation resulted in significant tumor regression, as in comparison with 5-FC or radiation treatments alone. Conclusions: Our data suggest that the hypoxia-inducible CDUPRT/5-FC gene therapy strategy has the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.

  4. Effect of albumin and dextrose concentration on ultrasound and microbubble mediated gene transfection in vivo.

    Science.gov (United States)

    Browning, Richard J; Mulvana, Helen; Tang, Meng-Xing; Hajnal, Jo V; Wells, Dominic J; Eckersley, Robert J

    2012-06-01

    Ultrasound and microbubble mediated gene transfection has great potential for site-selective, safe gene delivery. Albumin-based microbubbles have shown the greatest transfection efficiency but have not been optimised specifically for this purpose. Additionally, few studies have highlighted desirable properties for transfection specific microbubbles. In this article, microbubbles were made with 2% or 5% (w/v) albumin and 20% or 40% (w/v) dextrose solutions, yielding four distinct bubble types. These were acoustically characterised and their efficiency in transfecting a luciferase plasmid (pGL4.13) into female, CD1 mice myocardia was measured. For either albumin concentration, increasing the dextrose concentration increased scattering, attenuation and resistance to ultrasound, resulting in significantly increased transfection. A significant interaction was noted between albumin and dextrose; 2% albumin bubbles made with 20% dextrose showed the least transfection but the most transfection with 40% dextrose. This trend was seen for both nonlinear scattering and attenuation behaviour but not for resistance to ultrasound or total scatter. We have determined that the attenuation behaviour is an important microbubble characteristic for effective gene transfection using ultrasound. Microbubble behaviour can also be simply controlled by altering the initial ingredients used during manufacture. Copyright © 2012 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  5. Common Variation in the DOPA Decarboxylase (DDC) Gene and Human Striatal DDC Activity In Vivo.

    Science.gov (United States)

    Eisenberg, Daniel P; Kohn, Philip D; Hegarty, Catherine E; Ianni, Angela M; Kolachana, Bhaskar; Gregory, Michael D; Masdeu, Joseph C; Berman, Karen F

    2016-08-01

    The synthesis of multiple amine neurotransmitters, such as dopamine, norepinephrine, serotonin, and trace amines, relies in part on DOPA decarboxylase (DDC, AADC), an enzyme that is required for normative neural operations. Because rare, loss-of-function mutations in the DDC gene result in severe enzymatic deficiency and devastating autonomic, motor, and cognitive impairment, DDC common genetic polymorphisms have been proposed as a source of more moderate, but clinically important, alterations in DDC function that may contribute to risk, course, or treatment response in complex, heritable neuropsychiatric illnesses. However, a direct link between common genetic variation in DDC and DDC activity in the living human brain has never been established. We therefore tested for this association by conducting extensive genotyping across the DDC gene in a large cohort of 120 healthy individuals, for whom DDC activity was then quantified with [(18)F]-FDOPA positron emission tomography (PET). The specific uptake constant, Ki, a measure of DDC activity, was estimated for striatal regions of interest and found to be predicted by one of five tested haplotypes, particularly in the ventral striatum. These data provide evidence for cis-acting, functional common polymorphisms in the DDC gene and support future work to determine whether such variation might meaningfully contribute to DDC-mediated neural processes relevant to neuropsychiatric illness and treatment.

  6. Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection.

    Science.gov (United States)

    Debowski, Aleksandra W; Walton, Senta M; Chua, Eng-Guan; Tay, Alfred Chin-Yen; Liao, Tingting; Lamichhane, Binit; Himbeck, Robyn; Stubbs, Keith A; Marshall, Barry J; Fulurija, Alma; Benghezal, Mohammed

    2017-06-01

    Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress.

  7. Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection.

    Directory of Open Access Journals (Sweden)

    Aleksandra W Debowski

    2017-06-01

    Full Text Available Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress.

  8. Pharmacokinetics of gene recombined angiogenesis inhibitor Kringle 5 in vivo using 131I specific markers and SPECT/CT

    Directory of Open Access Journals (Sweden)

    Ge Yan

    2016-10-01

    Full Text Available The previous pharmacokinetic methods can be only limited to drug analysis in vitro, which provide less information on the distribution and metabolismof drugs, and limit the interpretation and assessment of pharmacokinetics, the determination of metabolic principles, and evaluation of treatment effect. The objective of the study was to investigate the pharmacokinetic characteristics of gene recombination angiogenesis inhibitor Kringle 5 in vivo. The SPECT/CT and specific 131I-Kringle 5 marked by Iodogen method were both applied to explore the pharmacokinetic characteristics of 131I-Kringle 5 in vivo, and to investigate the dynamic distributions of 131I-Kringle 5 in target organs. Labeling recombinant angiogenesis inhibitor Kringle 5 using 131I with longer half-life and imaging in vivo using SPECT instead of PET, could overcome the limitations of previous methods. When the doses of 131I-Kringle 5 were 10.0, 7.5 and 5.0 g/kg, respectively, the two-compartment open models can be determined within all the metabolic process in vivo. There were no significant differences in t1/2α, t1/2β, apparent volume of distribution and CL between those three levels. The ratio of AUC(0~∞ among three different groups of 10.0, 7.5 and 5.0 g/kg was 2.56:1.44:1.0, which was close to the ratio (2:1.5:1.0. It could be clear that in the range of 5.0–10.0 g/kg, Kringle 5 was characterized by the first-order pharmacokinetics. Approximately 30 min after 131I-Kringle 5 was injected, 131I-Kringle 5 could be observed to concentrate in the heart, kidneys, liver and other organs by means of planar imaging and tomography. After 1 h of being injected, more radionuclide retained in the bladder, but not in intestinal. It could be concluded that 131I-Kringle 5 is mainly excreted through the kidneys. About 2 h after the injection of 131I-Kringle 5, the radionuclide in the heart, kidneys, liver and other organs was gradually reduced, while more radionuclide was concentrated

  9. In vivo comparison of IVDU and IVFRU in HSV1-TK gene expressing tumor bearing rats

    Energy Technology Data Exchange (ETDEWEB)

    Choi, T.H.Tae Hyun; Ahn, S.H.Soon Hyuk; Kwon, H.-C.Hee-Chung; Choi, C.W.Chang Woon; Awh, O.D.Ok Doo; Lim, S.M.Sang Moo. E-mail: smlim328@kcch.re.kr

    2004-01-01

    (E)-5-(2-Iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-iodovinyl)-2'-fluoro-2'-deoxyuridine (IVFRU) are potential substrates of Herpes Simplex Virus type 1thymidine kinase (HSV-TK). In the present study, cellular uptake of radioiodinated substrates was found to be low in wild type MCA cells, but high in HSV-TK gene expressing cells. The carrier-free substrates, in particular, showed higher cellular uptake than carrier-added compounds. Biodistribution showed that the %ID/g of the MCA-TK/MCA tumor ratio of IVDU injected at 1, 4, and 24 h were 1.1, 0.9 and 1.3, and those of IVFRU were 1.7, 1.7 and 1.8 respectively. Therefore, both IVDU and IVFRU could possibly be used as radiopharmaceuticals to evaluate reporter gene expression. However, IVFRU was more specific and stable than IVDU for selective non-invasive imaging of HSV-TK expression.

  10. A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma.

    Science.gov (United States)

    Söling, Ariane; Theiss, Christian; Jungmichel, Stephanie; Rainov, Nikolai G

    2004-08-04

    BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease.

  11. In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

    International Nuclear Information System (INIS)

    Haupt, Larisa M; Thompson, Erik W; Trezise, Ann EO; Irving, Rachel E; Irving, Michael G; Griffiths, Lyn R

    2006-01-01

    Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state. In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma. We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in

  12. Plasticity of human skeletal muscle: gene expression to in vivo function.

    Science.gov (United States)

    Harridge, Stephen D R

    2007-09-01

    Human skeletal muscle is a highly heterogeneous tissue, able to adapt to the different challenges that may be placed upon it. When overloaded, a muscle adapts by increasing its size and strength through satellite-cell-mediated mechanisms, whereby protein synthesis is increased and new nuclei are added to maintain the myonuclear domain. This process is regulated by an array of mechanical, hormonal and nutritional signals. Growth factors, such as insulin-like growth factor I (IGF-I) and testosterone, are potent anabolic agents, whilst myostatin acts as a negative regulator of muscle mass. Insulin-like growth factor I is unique in being able to stimulate both the proliferation and the differentiation of satellite cells and works as part of an important local repair and adaptive mechanism. Speed of movement, as characterized by maximal velocity of shortening (V(max)), is regulated primarily by the isoform of myosin heavy chain (MHC) contained within a muscle fibre. Human fibres can express three MHCs: MHC-I, -IIa and -IIx, in order of increasing V(max) and maximal power output. Training studies suggest that there is a subtle interplay between the MHC-IIa and -IIx isoforms, with the latter being downregulated by activity and upregulated by inactivity. However, switching between the two main isoforms appears to require significant challenges to a muscle. Upregulation of fast gene programs is caused by prolonged disuse, whilst upregulation of slow gene programs appears to require significant and prolonged activity. The potential mechanisms by which alterations in muscle composition are mediated are discussed. The implications in terms of contractile function of altering muscle phenotype are discussed from the single fibre to the whole muscle level.

  13. Cationic liposome-mediated gene transfer to tumor cells in vitro and in vivo.

    Science.gov (United States)

    Son, K; Sorgi, F; Gao, X; Huang, L

    1997-01-01

    Development of safe and effective technology for delivering functional DNA into cells in an intact organism is crucial to broad applications of gene therapy to human disease. Both viral and nonviral vectors have been developed. Of the technologies currently being studied, liposomal delivery system is particularly attractive. Cationic liposome-mediated gene transfection (lipofection), a relatively new technique pioneered by Felgner and coworkers (1), was highly efficient for transfecting cells in culture. The liposomes were composed of an equimolar mixture of a synthetic cationic lipid N-[1-(2,3,-dioleyloxy)propyl]-N,N,N,-trimethylammonium chloride (DOTMA) and a helper lipid dioleoyl-phosphatidylethanolamine (DOPE) Fig. 1). The DOTMA/DOPE mixture (Lipofectin) forms complexes with DNA by charge interaction upon mixing at room temperature. Other catronic lipids are DOTAP, LipofectAMINE, Lipofectam, and DC-chol. The DOTAP is a diester analog of DOTMA and commercially available. LipofectAMINE and Lipofectam are polycationic lipids with a spermine head group that show increased frequency and activity of eukaryotic cell transfection (2,3). 3β-[N-(N',N'-dimethyaminoaminoethane) carbamoyl] cholesterol (DC-chol) (Fig. 1), a cationic cholesterol derivative, was introduced by Gao and Huang (4) and is routinely used in our laboratory. The DC-chol is now commercially available but can be easily synthesized with a single-step reaction from N,N-dimethylethylenediamine and cholesterol chloroformate (4), and improves the efficiency of transfection with minimal toxicity.Liposomes prepared with DC-chol and DOPE (3∶2 molar ratio) are stable at 4°C for at least 1 yr (unpublished data).

  14. In vivo gene transfer using pDNA/chitosan/chondroitin sulfate ternary complexes: influence of chondroitin sulfate on the stability of freeze-dried complexes and transgene expression in vivo.

    Science.gov (United States)

    Hagiwara, Kenji; Kishimoto, Satoko; Ishihara, Masayuki; Koyama, Yoshiyuki; Mazda, Osam; Sato, Toshinori

    2013-02-01

    Chitosan has been investigated as a promising nonviral vector. However, several problems still remain, such as a relatively low transfection efficiency and instability under physiological conditions. We previously demonstrated that a chondroitin sulfate (CS) coating enhanced the transfection efficiency and physicochemical stability of plasmid DNA (pDNA)/chitosan complexes in vitro. In the present study, the effects of coating pDNA/chitosan complexes with CS on the stability in freeze-dry rehydration processes and gene expression in vivo were investigated. Freeze-drying storage at -20 °C, 4 °C, or room temperature, freezing storage at -20 °C, or liquid storage at 4 °C or room temperature, were examined for preservation conditions of pDNA/chitosan/CS ternary complexes by a gel retardation assay, measurements of sizes and zeta potentials, and a luciferase assay. Moreover, to determine the transfection efficiency of the ternary complexes in vivo, suicide gene therapy was carried out in Huh-7-implanted mice using herpes simplex virus thymidine kinase coding pDNA and ganciclovir. The freeze-dried pDNA/chitosan/CS ternary complexes showed sufficient cell transfection ability in vitro and in vivo. In addition, ternary complexes were associated with a significant suppression of tumor growth and a histopathologically high anti-tumor effect by intratumoral injection to tumor-bearing mice. The CS coating enhanced the preservation stability of the pDNA/chitosan complexes after freeze-drying-rehydration and their transgene expression in vivo. Copyright © 2013 John Wiley & Sons, Ltd.

  15. A Convenient In Vivo Model Using Small Interfering RNA Silencing to Rapidly Assess Skeletal Gene Function.

    Directory of Open Access Journals (Sweden)

    Wen Zhang

    Full Text Available It is difficult to study bone in vitro because it contains various cell types that engage in cross-talk. Bone biologically links various organs, and it has thus become increasingly evident that skeletal physiology must be studied in an integrative manner in an intact animal. We developed a model using local intraosseous small interfering RNA (siRNA injection to rapidly assess the effects of a target gene on the local skeletal environment. In this model, 160-g male Sprague-Dawley rats were treated for 1-2 weeks. The left tibia received intraosseous injection of a parathyroid hormone 1 receptor (Pth1r or insulin-like growth factor 1 receptor (Igf-1r siRNA transfection complex loaded in poloxamer 407 hydrogel, and the right tibia received the same volume of control siRNA. All the tibias received an intraosseous injection of recombinant human parathyroid hormone (1-34 (rhPTH (1-34 or insulin-like growth factor-1 (IGF-1. Calcein green and alizarin red were injected 6 and 2 days before euthanasia, respectively. IGF-1R and PTH1R expression levels were detected via RT-PCR assays and immunohistochemistry. Bone mineral density (BMD, microstructure, mineral apposition rates (MARs, and strength were determined by dual-energy X-ray absorptiometry, micro-CT, histology and biomechanical tests. The RT-PCR and immunohistochemistry results revealed that IGF-1R and PTH1R expression levels were dramatically diminished in the siRNA-treated left tibias compared to the right tibias (both p<0.05. Using poloxamer 407 hydrogel as a controlled-release system prolonged the silencing effect of a single dose of siRNA; the mRNA expression levels of IGF-1R were lower at two weeks than at one week (p<0.01. The BMD, bone microstructure parameters, MAR and bone strength were significantly decreased in the left tibias compared to the right tibias (all p<0.05. This simple and convenient local intraosseous siRNA injection model achieved gene silencing with very small quantities of

  16. Influenza virus gene expression: viral RNA replication in vivo and in vitro

    International Nuclear Information System (INIS)

    Shapiro, G.I.

    1987-01-01

    To develop an overall scheme for the control of influenza virus gene expression, single-stranded M13 DNAs specific for the various genomic segments were used to analyze the synthesis of virus-specific RNAs in infected cells. The results showed that virus infection is divided into two distinct phases. During the early phase, the syntheses of specific virion RNAs (vRNAs), viral mRNAs, and viral proteins were coupled. This phase lasted for 2.5 hours in BHK-21 cells, the time when the rate of synthesis of all the viral mRNAs was maximal. During the late phase, the synthesis of all the vRNAs remained at or near maximum, whereas the rate of synthesis of all the viral mRNAs declined dramatically. Viral mRNA and protein syntheses were also not coupled, as the synthesis of all the viral proteins continued at maximum levels, indicating that protein synthesis during this phase was directed principally by previously synthesized viral mRNAs. Pulses with [ 3 H]uridine and nonaqueous fractionation of cells were used to show that influenza vRNA, like viral mRNAs, are synthesized in the nucleus and efficiently transported to the cytoplasm. In contrast, the full-length transcripts of the vRNAs, the templates for new vRNA synthesis, were synthesized only at early times, and remained sequestered in the nucleus to direct vRNA synthesis throughout infection

  17. Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins

    Science.gov (United States)

    Beissert, Tim; Koste, Lars; Perkovic, Mario; Walzer, Kerstin C.; Erbar, Stephanie; Selmi, Abderraouf; Diken, Mustafa; Kreiter, Sebastian; Türeci, Özlem; Sahin, Ugur

    2017-01-01

    Among nucleic acid–based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-β upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned. PMID:28877647

  18. mRNA levels of imprinted genes in bovine in vivo oocytes, embryos and cross species comparisons in humans, mice and pigs

    Science.gov (United States)

    Twenty-six confirmed imprinted genes in the bovine were quantified in in vivo produced oocytes and embryos. Eighteen were detectable and their transcriptional abundance were categorized into five patterns: largely decreased (MEST and PLAGL1); first decreased and then increased (CDKN1C and IGF2R); p...

  19. In vivo corrosion, tumor outcome, and microarray gene expression for two types of muscle-implanted tungsten alloys

    International Nuclear Information System (INIS)

    Schuster, B.E.; Roszell, L.E.; Murr, L.E.; Ramirez, D.A.; Demaree, J.D.; Klotz, B.R.; Rosencrance, A.B.; Dennis, W.E.; Bao, W.; Perkins, E.J.; Dillman, J.F.; Bannon, D.I.

    2012-01-01

    Tungsten alloys are composed of tungsten microparticles embedded in a solid matrix of transition metals such as nickel, cobalt, or iron. To understand the toxicology of these alloys, male F344 rats were intramuscularly implanted with pellets of tungsten/nickel/cobalt, tungsten/nickel/iron, or pure tungsten, with tantalum pellets as a negative control. Between 6 and 12 months, aggressive rhabdomyosarcomas formed around tungsten/nickel/cobalt pellets, while those of tungsten/nickel/iron or pure tungsten did not cause cancers. Electron microscopy showed a progressive corrosion of the matrix phase of tungsten/nickel/cobalt pellets over 6 months, accompanied by high urinary concentrations of nickel and cobalt. In contrast, non-carcinogenic tungsten/nickel/iron pellets were minimally corroded and urinary metals were low; these pellets having developed a surface oxide layer in vivo that may have restricted the mobilization of carcinogenic nickel. Microarray analysis of tumors revealed large changes in gene expression compared with normal muscle, with biological processes involving the cell cycle significantly up‐regulated and those involved with muscle development and differentiation significantly down‐regulated. Top KEGG pathways disrupted were adherens junction, p53 signaling, and the cell cycle. Chromosomal enrichment analysis of genes showed a highly significant impact at cytoband 7q22 (chromosome 7) which included mouse double minute (MDM2) and cyclin‐dependant kinase (CDK4) as well as other genes associated with human sarcomas. In conclusion, the tumorigenic potential of implanted tungsten alloys is related to mobilization of carcinogenic metals nickel and cobalt from corroding pellets, while gene expression changes in the consequent tumors are similar to radiation induced animal sarcomas as well as sporadic human sarcomas. -- Highlights: ► Tungsten/nickel/cobalt, tungsten/nickel/iron, and pure tungsten were studied. ► Male Fischer rats implanted with

  20. Mercury-induced hepatotoxicity in zebrafish: in vivo mechanistic insights from transcriptome analysis, phenotype anchoring and targeted gene expression validation

    Directory of Open Access Journals (Sweden)

    Mathavan Sinnakaruppan

    2010-03-01

    Full Text Available Abstract Background Mercury is a prominent environmental contaminant that causes detrimental effects to human health. Although the liver has been known to be a main target organ, there is limited information on in vivo molecular mechanism of mercury-induced toxicity in the liver. By using transcriptome analysis, phenotypic anchoring and validation of targeted gene expression in zebrafish, mercury-induced hepatotoxicity was investigated and a number of perturbed cellular processes were identified and compared with those captured in the in vitro human cell line studies. Results Hepato-transcriptome analysis of mercury-exposed zebrafish revealed that the earliest deregulated genes were associated with electron transport chain, mitochondrial fatty acid beta-oxidation, nuclear receptor signaling and apoptotic pathway, followed by complement system and proteasome pathway, and thereafter DNA damage, hypoxia, Wnt signaling, fatty acid synthesis, gluconeogenesis, cell cycle and motility. Comparative meta-analysis of microarray data between zebrafish liver and human HepG2 cells exposed to mercury identified some common toxicological effects of mercury-induced hepatotoxicity in both models. Histological analyses of liver from mercury-exposed fish revealed morphological changes of liver parenchyma, decreased nucleated cell count, increased lipid vesicles, glycogen and apoptotic bodies, thus providing phenotypic evidence for anchoring of the transcriptome analysis. Validation of targeted gene expression confirmed deregulated gene-pathways from enrichment analysis. Some of these genes responding to low concentrations of mercury may serve as toxicogenomic-based markers for detection and health risk assessment of environmental mercury contaminations. Conclusion Mercury-induced hepatotoxicity was triggered by oxidative stresses, intrinsic apoptotic pathway, deregulation of nuclear receptor and kinase activities including Gsk3 that deregulates Wnt signaling

  1. In vivo corrosion, tumor outcome, and microarray gene expression for two types of muscle-implanted tungsten alloys

    Energy Technology Data Exchange (ETDEWEB)

    Schuster, B.E. [U.S. Army Research Laboratory, Weapons and Materials Research Directorate, B434 Mulberry Road, Aberdeen Proving Ground, MD 21005-5609 (United States); Roszell, L.E. [U.S. Army Institute of Public Health, 5158 Blackhawk Road, Aberdeen Proving Ground, MD 21010‐5403 (United States); Murr, L.E.; Ramirez, D.A. [Department of Metallurgical and Materials Engineering, University of Texas, El Paso, TX 79968 (United States); Demaree, J.D. [U.S. Army Research Laboratory, Weapons and Materials Research Directorate, B434 Mulberry Road, Aberdeen Proving Ground, MD 21005-5609 (United States); Klotz, B.R. [Dynamic Science Inc., Aberdeen Proving Ground, MD 21005‐5609 (United States); Rosencrance, A.B.; Dennis, W.E. [U.S. Army Center for Environmental Health Research, Department of Chemistry, Ft. Detrick, MD 21702‐5010 (United States); Bao, W. [SAS Institute, Inc. SAS Campus Drive, Cary, NC 27513 (United States); Perkins, E.J. [U.S. Army Engineer Research and Development Center, 3909 Hall Ferry Road, Vicksburg MS 39180 (United States); Dillman, J.F. [U.S. Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, MD 21010‐5400 (United States); Bannon, D.I., E-mail: desmond.bannon@us.army.mil [U.S. Army Institute of Public Health, 5158 Blackhawk Road, Aberdeen Proving Ground, MD 21010‐5403 (United States)

    2012-11-15

    Tungsten alloys are composed of tungsten microparticles embedded in a solid matrix of transition metals such as nickel, cobalt, or iron. To understand the toxicology of these alloys, male F344 rats were intramuscularly implanted with pellets of tungsten/nickel/cobalt, tungsten/nickel/iron, or pure tungsten, with tantalum pellets as a negative control. Between 6 and 12 months, aggressive rhabdomyosarcomas formed around tungsten/nickel/cobalt pellets, while those of tungsten/nickel/iron or pure tungsten did not cause cancers. Electron microscopy showed a progressive corrosion of the matrix phase of tungsten/nickel/cobalt pellets over 6 months, accompanied by high urinary concentrations of nickel and cobalt. In contrast, non-carcinogenic tungsten/nickel/iron pellets were minimally corroded and urinary metals were low; these pellets having developed a surface oxide layer in vivo that may have restricted the mobilization of carcinogenic nickel. Microarray analysis of tumors revealed large changes in gene expression compared with normal muscle, with biological processes involving the cell cycle significantly up‐regulated and those involved with muscle development and differentiation significantly down‐regulated. Top KEGG pathways disrupted were adherens junction, p53 signaling, and the cell cycle. Chromosomal enrichment analysis of genes showed a highly significant impact at cytoband 7q22 (chromosome 7) which included mouse double minute (MDM2) and cyclin‐dependant kinase (CDK4) as well as other genes associated with human sarcomas. In conclusion, the tumorigenic potential of implanted tungsten alloys is related to mobilization of carcinogenic metals nickel and cobalt from corroding pellets, while gene expression changes in the consequent tumors are similar to radiation induced animal sarcomas as well as sporadic human sarcomas. -- Highlights: ► Tungsten/nickel/cobalt, tungsten/nickel/iron, and pure tungsten were studied. ► Male Fischer rats implanted with

  2. Targeted gene delivery to the synovial pannus in antigen-induced arthritis by ultrasound-targeted microbubble destruction in vivo.

    Science.gov (United States)

    Xiang, Xi; Tang, Yuanjiao; Leng, Qianying; Zhang, Lingyan; Qiu, Li

    2016-02-01

    The purpose of this study was to optimize an ultrasound-targeted microbubble destruction (UTMD) technique to improve the in vivo transfection efficiency of the gene encoding enhanced green fluorescent protein (EGFP) in the synovial pannus in an antigen-induced arthritis rabbit model. A mixture of microbubbles and plasmids was locally injected into the knee joints of an antigen-induced arthritis (AIA) rabbits. The plasmid concentrations and ultrasound conditions were varied in the experiments. We also tested local articular and intravenous injections. The rabbits were divided into five groups: (1) ultrasound+microbubbles+plasmid; (2) ultrasound+plasmid; (3) microbubble+plasmid; (4) plasmid only; (5) untreated controls. EGFP expression was observed by fluorescent microscope and immunohistochemical staining in the synovial pannus of each group. The optimal plasmid dosage and ultrasound parameter were determined based on the results of EGFP expression and the present and absent of tissue damage under light microscopy. The irradiation procedure was performed to observe the duration of the EGFP expression in the synovial pannus and other tissues and organs, as well as the damage to the normal cells. The optimal condition was determined to be a 1-MHz ultrasound pulse applied for 5 min with a power output of 2 W/cm(2) and a 20% duty cycle along with 300 μg of plasmid. Under these conditions, the synovial pannus showed significant EGFP expression without significant damage to the surrounding normal tissue. The EGFP expression induced by the local intra-articular injection was significantly more increased than that induced by the intravenous injection. The EGFP expression in the synovial pannus of the ultrasound+microbubbles+plasmid group was significantly higher than that of the other four groups (Ppannus of an AIA model. Thus, this could become a safe and effective non-viral gene transfection procedure for arthritis therapy. Copyright © 2015 Elsevier B.V. All rights

  3. The (PrS/HGF-pDNA) multilayer films for gene-eluting stent coating: Gene-protecting, anticoagulation, antibacterial properties, and in vivo antirestenosis evaluation.

    Science.gov (United States)

    Chang, Hao; Ren, Ke-feng; Zhang, He; Wang, Jin-lei; Wang, Bai-liang; Ji, Jian

    2015-02-01

    Vascular gene-eluting stents (GES) is a promising strategy for treatment of cardiovascular disease. Very recently, we have proved that the (protamine sulfate/plasmid DNA encoding hepatocyte growth factor) (PrS/HGF-pDNA) multilayer can serve as a powerful tool for enhancing competitiveness of endothelial cell over smooth muscle cell, which opens perspectives for the regulation of intercellular competitiveness in the field of interventional therapy. However, before the gene multilayer films could be used in vascular stents for real clinical application, the preservation of gene bioactivity during the industrial sterilization and the hemocompatibility of film should be taken into account. Actually, both are long been ignored issues in the field of gene coating for GES. In this study, we demonstrate that the (PrS/HGF-pDNA) multilayer film exhibits the good gene-protecting abilities, which is confirmed by using the industrial sterilizations (gamma irradiation and ethylene oxide) and a routine storage condition (dry state at 4°C for 30 days). Furthermore, hemocompatible measurements (such as platelet adhesion and whole blood coagulation) and antibacterial assays (bacteria adhesion and growth inhibition) indicate the good anticoagulation and antibacterial properties of the (PrS/HGF-pDNA) multilayer film. The in vivo preliminary data of angiography and histological analysis suggest that the (PrS/HGF-pDNA) multilayer coated stent can reduce the in-stent restenosis. This work reveals that the (PrS/HGF-pDNA) multilayer film could be a promising candidate as coating for GES, which is of great potential in future clinic application. © 2014 Wiley Periodicals, Inc.

  4. Telomeric repeat-containing RNA/G-quadruplex-forming sequences cause genome-wide alteration of gene expression in human cancer cells in vivo.

    Science.gov (United States)

    Hirashima, Kyotaro; Seimiya, Hiroyuki

    2015-02-27

    Telomere erosion causes cell mortality, suggesting that longer telomeres enable more cell divisions. In telomerase-positive human cancer cells, however, telomeres are often kept shorter than those of surrounding normal tissues. Recently, we showed that cancer cell telomere elongation represses innate immune genes and promotes their differentiation in vivo. This implies that short telomeres contribute to cancer malignancy, but it is unclear how such genetic repression is caused by elongated telomeres. Here, we report that telomeric repeat-containing RNA (TERRA) induces a genome-wide alteration of gene expression in telomere-elongated cancer cells. Using three different cell lines, we found that telomere elongation up-regulates TERRA signal and down-regulates innate immune genes such as STAT1, ISG15 and OAS3 in vivo. Ectopic TERRA oligonucleotides repressed these genes even in cells with short telomeres under three-dimensional culture conditions. This appeared to occur from the action of G-quadruplexes (G4) in TERRA, because control oligonucleotides had no effect and a nontelomeric G4-forming oligonucleotide phenocopied the TERRA oligonucleotide. Telomere elongation and G4-forming oligonucleotides showed similar gene expression signatures. Most of the commonly suppressed genes were involved in the innate immune system and were up-regulated in various cancers. We propose that TERRA G4 counteracts cancer malignancy by suppressing innate immune genes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. In vivo imaging of herpes simplex virus type 1 thymidine kinase gene expression: early kinetics of radiolabelled FIAU

    Energy Technology Data Exchange (ETDEWEB)

    Haubner, R.; Avril, N.; Schwaiger, M. [Technische Univ. Muenchen (Germany). Nuklearmedizinische Klinik und Poliklinik; Hantzopoulos, P.A.; Gansbacher, B. [Inst. of Experimental Oncology, Technische Univ. Muenchen (Germany)

    2000-03-01

    The aim of our study was to examine the early kinetics of I*-FIAU and the possibility of utilising iodine-123-labelled FIAU for imaging of gene expression. CMS-5 fibrosarcoma cells were transduced in vitro with the retroviral vector STK containing the HSV1-tk gene. BALB/c mice were inoculated subcutaneously with HSV1-tk(+) and tk(-) cells into both flanks. FAU (2'-fluoro-2'-deoxy-1-{beta}-d-arabinofuranosyluracil was radioiodinated ({sup 123}I, {sup 125}I)) using the iodogen method. High-performance liquid chromatography purification resulted in high specific activity and radiochemical purity for both tracers ([{sup 123}I]FIAU and [{sup 125}I]FIAU). Biodistribution studies and gamma camera imaging were performed at 0.5, 1, 2 and 4 h p.i. In addition, the genomic DNA of the tumours was isolated for measurement of the activity accumulation resulting from the [{sup 125}I]FIAU incorporation. Biodistribution studies 0.5 h p.i. showed tumour/blood and tumour/muscle ratios of 3.8 and 7.2, respectively, for the HSV1-tk(+) tumours, and 0.6 and 1.2, respectively, for negative control tumours. Fast renal elimination of the tracer from the body resulted in rapidly increasing tumour/blood and tumour/muscle ratios which reached values of 32 and 88 at 4 h p.i., respectively. Tracer clearance from blood was bi-exponential, with an initial half-life of 0.6 h followed by a half-life of 4.6 h. The tracer half-life in herpes simplex viral thymidine kinase-expressing tumours was 35.7 h. The highest activity accumulation (20.3%{+-}5.7% ID/g) in HSV1-tk(+) tumours was observed 1 h p.i. At that time, about 46% of the total activity found in HSV1-tk(+) tumours was incorporated into genomic DNA. Planar gamma camera imaging showed a distinct tracer accumulation as early as 0.5 h p.i., with an increase in contrast over time. These results suggest that sufficient tumour/background ratios for in vivo imaging of HSV1-tk expression with [{sup 123}I]FIAU are reached as early as 1 h p

  6. In vivo imaging of induction of heat-shock protein-70 gene expression with fluorescence reflectance imaging and intravital confocal microscopy following brain ischaemia in reporter mice.

    Science.gov (United States)

    de la Rosa, Xavier; Santalucía, Tomàs; Fortin, Pierre-Yves; Purroy, Jesús; Calvo, Maria; Salas-Perdomo, Angélica; Justicia, Carles; Couillaud, Franck; Planas, Anna M

    2013-02-01

    Stroke induces strong expression of the 72-kDa heat-shock protein (HSP-70) in the ischaemic brain, and neuronal expression of HSP-70 is associated with the ischaemic penumbra. The aim of this study was to image induction of Hsp-70 gene expression in vivo after brain ischaemia using reporter mice. A genomic DNA sequence of the Hspa1b promoter was used to generate an Hsp70-mPlum far-red fluorescence reporter vector. The construct was tested in cellular systems (NIH3T3 mouse fibroblast cell line) by transient transfection and examining mPlum and Hsp-70 induction under a challenge. After construct validation, mPlum transgenic mice were generated. Focal brain ischaemia was induced by transient intraluminal occlusion of the middle cerebral artery and the mice were imaged in vivo with fluorescence reflectance imaging (FRI) with an intact skull, and with confocal microscopy after opening a cranial window. Cells transfected with the Hsp70-mPlum construct showed mPlum fluorescence after stimulation. One day after induction of ischaemia, reporter mice showed a FRI signal located in the HSP-70-positive zone within the ipsilateral hemisphere, as validated by immunohistochemistry. Live confocal microscopy allowed brain tissue to be visualized at the cellular level. mPlum fluorescence was observed in vivo in the ipsilateral cortex 1 day after induction of ischaemia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction. This study showed in vivo induction of Hsp-70 gene expression in ischaemic brain using reporter mice. The fluorescence signal showed in vivo the induction of Hsp-70 in penumbra neurons and in the vasculature within the ischaemic core.

  7. Hypoxia induces telomerase reverse transcriptase (TERT gene expression in non-tumor fish tissues in vivo: the marine medaka (Oryzias melastigma model

    Directory of Open Access Journals (Sweden)

    Mok Helen OL

    2006-09-01

    Full Text Available Abstract Background Current understanding on the relationships between hypoxia, hypoxia-inducible factor-1 (HIF-1 and telomerase reverse transcriptase (TERT gene expression are largely based on in vitro studies in human cancer cells. Although several reports demonstrated HIF-1- mediated upregulation of the human TERT gene under hypoxia, conflicting findings have also been reported. Thus far, it remains uncertain whether these findings can be directly extrapolated to non-tumor tissues in other whole animal systems in vivo. While fish often encounter environmental hypoxia, the in vivo regulation of TERT by hypoxia in non-neoplastic tissues of fish remains virtually unknown. Results The adult marine medaka (Oryzias melastigma was employed as a model fish in this study. We have cloned and characterized a 3261-bp full-length TERT cDNA, omTERT, which encodes a protein of 1086 amino acids. It contains all of the functional motifs that are conserved in other vertebrate TERTs. Motif E is the most highly conserved showing 90.9–100% overall identity among the fish TERTs and 63.6% overall identity among vertebrates. Analysis of the 5'-flanking sequence of the omTERT gene identified two HRE (hypoxia-responsive element; nt. – 283 and – 892 cores. Overexpression of the HIF-1α induced omTERT promoter activity as demonstrated using transient transfection assays. The omTERT gene is ubiquitously expressed in fish under normoxia, albeit at varying levels, where highest expression was observed in gonads and the lowest in liver. In vivo expression of omTERT was significantly upregulated in testis and liver in response to hypoxia (at 96 h and 48 h, respectively, where concomitant induction of the omHIF-1α and erythropoietin (omEpo genes was also observed. In situ hybridization analysis showed that hypoxic induction of omTERT mRNA was clearly evident in hepatocytes in the caudal region of liver and in spermatogonia-containing cysts in testis. Conclusion This

  8. Tax gene expression and cell cycling but not cell death are selected during HTLV-1 infection in vivo

    Directory of Open Access Journals (Sweden)

    Pinatel Christiane

    2010-03-01

    Full Text Available Abstract Background Adult T cell leukemia results from the malignant transformation of a CD4+ lymphoid clone carrying an integrated HTLV-1 provirus that has undergone several oncogenic events over a 30-60 year period of persistent clonal expansion. Both CD4+ and CD8+ lymphocytes are infected in vivo; their expansion relies on CD4+ cell cycling and on the prevention of CD8+ cell death. Cloned infected CD4+ but not CD8+ T cells from patients without malignancy also add up nuclear and mitotic defects typical of genetic instability related to theexpression of the virus-encoded oncogene tax. HTLV-1 expression is cancer-prone in vitro, but in vivo numerous selection forces act to maintain T cell homeostasis and are possibly involved in clonal selection. Results Here we demonstrate that the HTLV-1 associated CD4+ preleukemic phenotype and the specific patterns of CD4+ and CD8+ clonal expansion are in vivo selected processes. By comparing the effects of recent (1 month experimental infections performed in vitro and those observed in cloned T cells from patients infected for >6-26 years, we found that in chronically HTLV-1 infected individuals, HTLV-1 positive clones are selected for tax expression. In vivo, infected CD4+ cells are positively selected for cell cycling whereas infected CD8+ cells and uninfected CD4+ cells are negatively selected for the same processes. In contrast, the known HTLV-1-dependent prevention of CD8+ T cell death pertains to both in vivo and in vitro infected cells. Conclusions Therefore, virus-cell interactions alone are not sufficient to initiate early leukemogenesis in vivo.

  9. In vitro radionuclide therapy and in vivo scintigraphic imaging of alpha fetoprotein producing hepatocellular carcinoma by targeted sodium iodide symporter gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kwang Il; Lee, Yong Jin; Lee, Tae Sup; Song, Inho; Cheon, Gi Jeong; Lim, Sang Moo; Kang, Joo Hyun [Korea Institute of Radiological and Medical and Medical Sciences, Seoul (Korea, Republic of); Chung, June Key [Seoul National Univ. College of Medicine, Seoul (Korea, Republic of)

    2012-03-15

    This study aimed to develop a gene expression targeting method for specific imaging and therapy of alpha fetoprotein (AFP) producing hepatocellular carcinoma (HCC) cells, using an adenovirus vector containing the human sodium/iodide symporter (hNIS) gene driven by an AFP enhancer/promoter. The recombinant adenovirus vector, AdAFPhNIS (containing the hNIS gene driven by human AFP enhancer/promoter) was prepared. After in vitro infection by the adenovirus, hNIS gene expression in AFP producing cells and in AFP nonproducing cells was investigated using {sup 125}I uptake assay and semi quantitative reverse transcription polymerase chain reaction (RT-PCR). The killing effect of {sup 131}I vitro clonogenic assay. In addition, tumor bearing mice were intravenously injected with the adenovirus, and scintigraphic images were obtained. The expression of hNIS was efficiently demonstrated by {sup 125}I uptake assay in AFP producing cells, but not in AFP nonproducing cells. AFP producing HCC targeted gene expression was confirmed at the mRNA level. Furthermore, in vitro clonogenic assay showed that hNIS gene expression induced by AdAFPhNIS infection in AFP producing cells caused more sensitivity to {sup 131}I than that in AFP nonproducing cells. Injected intravenously in HuH-7 tumor xenografts mice by adenovirus, the functional hNIS gene expression was confirmed in tumor by in vivo scintigraphic imaging. An AFP producing HCC was targeted with an adenovirus vector containing the hNIS gene using the AFP enhancer/promoter in vitro and in vivo. These findings demonstrate that AFP producing HCC specific molecular imaging and radionuclide gene therapy are feasible using this recombinant adenovirus vector system.

  10. In vitro radionuclide therapy and in vivo scintigraphic imaging of alpha fetoprotein producing hepatocellular carcinoma by targeted sodium iodide symporter gene expression

    International Nuclear Information System (INIS)

    Kim, Kwang Il; Lee, Yong Jin; Lee, Tae Sup; Song, Inho; Cheon, Gi Jeong; Lim, Sang Moo; Kang, Joo Hyun; Chung, June Key

    2012-01-01

    This study aimed to develop a gene expression targeting method for specific imaging and therapy of alpha fetoprotein (AFP) producing hepatocellular carcinoma (HCC) cells, using an adenovirus vector containing the human sodium/iodide symporter (hNIS) gene driven by an AFP enhancer/promoter. The recombinant adenovirus vector, AdAFPhNIS (containing the hNIS gene driven by human AFP enhancer/promoter) was prepared. After in vitro infection by the adenovirus, hNIS gene expression in AFP producing cells and in AFP nonproducing cells was investigated using 125 I uptake assay and semi quantitative reverse transcription polymerase chain reaction (RT-PCR). The killing effect of 131 I vitro clonogenic assay. In addition, tumor bearing mice were intravenously injected with the adenovirus, and scintigraphic images were obtained. The expression of hNIS was efficiently demonstrated by 125 I uptake assay in AFP producing cells, but not in AFP nonproducing cells. AFP producing HCC targeted gene expression was confirmed at the mRNA level. Furthermore, in vitro clonogenic assay showed that hNIS gene expression induced by AdAFPhNIS infection in AFP producing cells caused more sensitivity to 131 I than that in AFP nonproducing cells. Injected intravenously in HuH-7 tumor xenografts mice by adenovirus, the functional hNIS gene expression was confirmed in tumor by in vivo scintigraphic imaging. An AFP producing HCC was targeted with an adenovirus vector containing the hNIS gene using the AFP enhancer/promoter in vitro and in vivo. These findings demonstrate that AFP producing HCC specific molecular imaging and radionuclide gene therapy are feasible using this recombinant adenovirus vector system

  11. mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages.

    Science.gov (United States)

    Danesh Mesgaran, Sadjad; Sharbati, Jutta; Einspanier, Ralf; Gabler, Christoph

    2016-08-15

    The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality. BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h. Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0. This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation

  12. Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo

    NARCIS (Netherlands)

    Pfeifer, A.; Brandon, E. P.; Kootstra, N.; Gage, F. H.; Verma, I. M.

    2001-01-01

    The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly, we found a significant reduction in proliferation and an accumulation in the G(2)/M

  13. Transfection mediated by pH-sensitive sugar-based gemini surfactants; potential for in vivo gene therapy applications

    NARCIS (Netherlands)

    Wasungu, Luc; Scarzello, Marco; van Dam, Gooitzen; Molema, Grietje; Wagenaar, Anno; Engberts, Jan B. F. N.; Hoekstra, Dick

    In this study, the in vitro and in vivo transfection capacity of novel pH-sensitive sugar-based gemini surfactants was investigated. In an aqueous environment at physiological pH, these compounds form bilayer vesicles, but they undergo a lamellar-to-micellar phase transition in the endosomal pH

  14. Horizontal transmission of malignancy: in-vivo fusion of human lymphomas with hamster stroma produces tumors retaining human genes and lymphoid pathology.

    Directory of Open Access Journals (Sweden)

    David M Goldenberg

    Full Text Available We report the in-vivo fusion of two Hodgkin lymphomas with golden hamster cheek pouch cells, resulting in serially-transplanted (over 5-6 years GW-532 and GW-584 heterosynkaryon tumor cells displaying both human and hamster DNA (by FISH, lymphoma-like morphology, aggressive metastasis, and retention of 7 human genes (CD74, CXCR4, CD19, CD20, CD71, CD79b, and VIM out of 24 tested by PCR. The prevalence of B-cell restricted genes (CD19, CD20, and CD79b suggests that this uniform population may be the clonal initiating (malignant cells of Hodgkin lymphoma, despite their not showing translation to their respective proteins by immunohistochemical analysis. This is believed to be the first report of in-vivo cell-cell fusion of human lymphoma and rodent host cells, and may be a method to disclose genes regulating both organoid and metastasis signatures, suggesting that the horizontal transfer of tumor DNA to adjacent stromal cells may be implicated in tumor heterogeneity and progression. The B-cell gene signature of the hybrid xenografts suggests that Hodgkin lymphoma, or its initiating cells, is a B-cell malignancy.

  15. In vitro and in vivo effects of polyethylene glycol (PEG)-modified lipid in DOTAP/cholesterol-mediated gene transfection

    DEFF Research Database (Denmark)

    Gjetting, Torben; Arildsen, Nicolai Skovbjerg; Christensen, Camilla Laulund

    2010-01-01

    DOTAP/cholesterol-based lipoplexes are successfully used for delivery of plasmid DNA in vivo especially to the lungs, although low systemic stability and circulation have been reported. To achieve the aim of discovering the best method for systemic delivery of DNA to disseminated tumors we evalua...... evaluated the potential of formulating DOTAP/cholesterol lipoplexes with a polyethylene glycol (PEG)-modified lipid, giving the benefit of the shielding and stabilizing properties of PEG in the bloodstream....

  16. Dioxin exposure of human CD34+ hemopoietic cells induces gene expression modulation that recapitulates its in vivo clinical and biological effects

    International Nuclear Information System (INIS)

    Fracchiolla, Nicola Stefano; Todoerti, Katia; Bertazzi, Pier Alberto; Servida, Federica; Corradini, Paolo; Carniti, Cristiana; Colombi, Antonio; Cecilia Pesatori, Angela; Neri, Antonino; Deliliers, Giorgio Lambertenghi

    2011-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has a large number of biological effects, including skin, cardiovascular, neurologic diseases, diabetes, infertility, cancers and immunotoxicity. We analysed the in vitro TCDD effects on human CD34 + cells and tested the gene expression modulation by means of microarray analyses before and after TCDD exposure. We identified 257 differentially modulated probe sets, identifying 221 well characterized genes. A large part of these resulted associated to cell adhesion and/or angiogenesis and to transcription regulation. Synaptic transmission and visual perception functions, with the particular involvement of the GABAergic pathway were also significantly modulated. Numerous transcripts involved in cell cycle or cell proliferation, immune response, signal transduction, ion channel activity or calcium ion binding, tissue development and differentiation, female or male fertility or in several metabolic pathways were also affected after dioxin exposure. The transcriptional profile induced by TCDD treatment on human CD34 + cells strikingly reproduces the clinical and biological effects observed in individuals exposed to dioxin and in biological experimental systems. Our data support a role of dioxin in the neoplastic transformation of hemopoietic stem cells and in immune modulation processes after in vivo exposure, as indicated by the epidemiologic data in dioxin accidentally exposed populations, providing a molecular basis for it. In addition, TCDD alters genes associated to glucidic and lipidic metabolisms, to GABAergic transmission or involved in male and female fertility, thus providing a possible explanation of the diabetogenic, dyslipidemic, neurologic and fertility effects induced by TCDD in vivo exposure.

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

    Energy Technology Data Exchange (ETDEWEB)

    Fornace, Jr, A J

    2007-03-03

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

  18. Pathological study on the testis of mice irradiated by γ-rays after transfecting pprI gene by in vivo electroporation

    International Nuclear Information System (INIS)

    Lian Lixia; Chen Tingting; Zhang Yongqin; Wang Xiuzhen; Yang Zhanshan

    2011-01-01

    To investigate the effects of pprI gene from Deinococcus radiodurans transferred by in vivo electroporation on γ-ray injury of mice, the morphological changes of testis in the mice were observed. The pCMV-HA-pprI plasmid containing pprI gene was injected into the muscle of mice. The pprI gene was transfected into the cells by in vivo gene electroporation technology. Then the control group and the transferred pCMV-HA-pprI group were exposed to γ-ray radiation of 6 Gy. The muscle tissue at the site of the injection and the testis tissue were taken on days 1, 7, 14, 28 and 35 after radiation. Then total protein was extracted and used to test the expression of PprI with western blotting technology. The testis specimen prepared by hematoxylin-eosin staining was then examined by light microscopy. The expression of PprI is remarkable on the 1 st day after irradiation to prove that the pprI gene was successfully transfected into the mice. On the 1 st day after irradiation there was no obvious pathological change of the testis tissue of the control group. On the 7th day there was degeneration and necrosis of some spermatogonia and spermatocytes in sections of tubules. On the 14th day, the reduction of spermatogonia was generally marked, and there was considerable reduction in the number of primary spermatocytes associated with atrophy of the seminiferous tubules. On the 28th day there was complete depletion of spermatogenic epithelium when spermatocytes and spermatids had largely disappeared, with no regeneration of spermatogonia and only sertoli cells nuclei remaining along the basement membrane. On the 35th day, spermatogonia were actively regenerating in some of the tubules. Compared with the control group, there was also no significant difference on the 1 st after irradiation in the transgenic animal. On the 7th day the degeneration and necrosis of some spermatogonia and spermatocytes in sections of tubules was less than that of the control group. On the 14th day the

  19. Minimal doses of a sequence-optimized transgene mediate high-level and long-term EPO expression in vivo: challenging CpG-free gene design.

    Science.gov (United States)

    Kosovac, D; Wild, J; Ludwig, C; Meissner, S; Bauer, A P; Wagner, R

    2011-02-01

    Advanced gene delivery techniques can be combined with rational gene design to further improve the efficiency of plasmid DNA (pDNA)-mediated transgene expression in vivo. Herein, we analyzed the influence of intragenic sequence modifications on transgene expression in vitro and in vivo using murine erythropoietin (mEPO) as a transgene model. A single electro-gene transfer of an RNA- and codon-optimized mEPOopt gene into skeletal muscle resulted in a 3- to 4-fold increase of mEPO production sustained for >1 year and triggered a significant increase in hematocrit and hemoglobin without causing adverse effects. mEPO expression and hematologic levels were significantly lower when using comparable amounts of the wild type (mEPOwt) gene and only marginal effects were induced by mEPOΔCpG lacking intragenic CpG dinucleotides, even at high pDNA amounts. Corresponding with these observations, in vitro analysis of transfected cells revealed a 2- to 3-fold increased (mEPOopt) and 50% decreased (mEPOΔCpG) erythropoietin expression compared with mEPOwt, respectively. RNA analyses demonstrated that the specific design of the transgene sequence influenced expression levels by modulating transcriptional activity and nuclear plus cytoplasmic RNA amounts rather than translation. In sum, whereas CpG depletion negatively interferes with efficient expression in postmitotic tissues, mEPOopt doses <0.5 μg were sufficient to trigger optimal long-term hematologic effects encouraging the use of sequence-optimized transgenes to further reduce effective pDNA amounts.

  20. Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo.

    Directory of Open Access Journals (Sweden)

    Keshab Rijal

    2016-08-01

    Full Text Available The ability of RNA polymerase (RNAP III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC; they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease.

  1. Selective in vivo radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells transduced with the E. coli cytosine deaminase (CD) gene

    International Nuclear Information System (INIS)

    Gabel, M.; Kim, J.H.; Kolozsvary, A.; Khil, M.; Freytag, S.

    1998-01-01

    Purpose: The E. coli cytosine deaminase (CD) gene encodes an enzyme capable of converting the nontoxic prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a known radiosensitizer. Having previously shown that combined CD suicide gene therapy and radiation (RT) results in pronounced radiosensitization in vitro, we progressed to in vivo studies of combined therapy. Methods and Materials: WiDr human colon cancer cells were transduced in vitro with the CD gene and cells expressing CD were selected for use as xenografts in a nude mouse model. After administration of 5-FC, tumors received 10-30 Gy local field radiation (RT) and tumor growth delay was compared to control animals receiving either 5-FU, 5-FC, or RT alone. Results: Maximal growth delay was seen in mice treated with 5-FC for 6 consecutive days prior to RT. Combined treatment with 15 Gy radiation resulted in a dose-modifying factor (DMF) of 1.50, and a greater DMF was observed with higher doses of radiation. There was no appreciable toxicity using this new approach. In contrast, a similar treatment of combined 5-FU and radiation resulted in considerable toxicity and no appreciable radiosensitization. Conclusion: The present results show that combined suicide gene therapy and RT results in pronounced antitumor effect without any notable toxicity. This indicates that the CD gene may be useful in the development of novel treatment strategies combining radiation and gene therapy in the treatment of locally advanced cancers

  2. Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice.

    Science.gov (United States)

    Sato, Masahiro; Inada, Emi; Saitoh, Issei; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

    2013-11-01

    The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ. © 2013 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptions are made.

  3. Genetic and biochemical evidence that haploinsufficiency of the Nf1 tumor suppressor gene modulates melanocyte and mast cell fates in vivo.

    Science.gov (United States)

    Ingram, D A; Yang, F C; Travers, J B; Wenning, M J; Hiatt, K; New, S; Hood, A; Shannon, K; Williams, D A; Clapp, D W

    2000-01-03

    Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's "two hit" model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1-/- murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W(41) mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras-mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W(41)) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types.

  4. Cold atmospheric plasma (CAP changes gene expression of key molecules of the wound healing machinery and improves wound healing in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Stephanie Arndt

    Full Text Available Cold atmospheric plasma (CAP has the potential to interact with tissue or cells leading to fast, painless and efficient disinfection and furthermore has positive effects on wound healing and tissue regeneration. For clinical implementation it is necessary to examine how CAP improves wound healing and which molecular changes occur after the CAP treatment. In the present study we used the second generation MicroPlaSter ß® in analogy to the current clinical standard (2 min treatment time in order to determine molecular changes induced by CAP using in vitro cell culture studies with human fibroblasts and an in vivo mouse skin wound healing model. Our in vitro analysis revealed that the CAP treatment induces the expression of important key genes crucial for the wound healing response like IL-6, IL-8, MCP-1, TGF-ß1, TGF-ß2, and promotes the production of collagen type I and alpha-SMA. Scratch wound healing assays showed improved cell migration, whereas cell proliferation analyzed by XTT method, and the apoptotic machinery analyzed by protein array technology, was not altered by CAP in dermal fibroblasts. An in vivo wound healing model confirmed that the CAP treatment affects above mentioned genes involved in wound healing, tissue injury and repair. Additionally, we observed that the CAP treatment improves wound healing in mice, no relevant side effects were detected. We suggest that improved wound healing might be due to the activation of a specified panel of cytokines and growth factors by CAP. In summary, our in vitro human and in vivo animal data suggest that the 2 min treatment with the MicroPlaSter ß® is an effective technique for activating wound healing relevant molecules in dermal fibroblasts leading to improved wound healing, whereas the mechanisms which contribute to these observed effects have to be further investigated.

  5. Analysis of clustered point mutations in the human ribosomal RNA gene promoter by transient expression in vivo

    International Nuclear Information System (INIS)

    Jones, M.H.; Learned, R.M.; Tjian, R.

    1988-01-01

    The authors have mapped the cis regulatory elements required in vivo for initiation at the human rRNA promoter by RNA polymerase I. Transient expression in COS-7 cells was used to evaluate the transcription phenotype of clustered base substitution mutations in the human rRNA promoter. The promoter consists of two major elements: a large upstream region, composed of several domains, that lies between nucleotides -234 and -107 relative to the transcription initiation site and affects transcription up to 100-fold and a core element that lies between nucleotides -45 and +20 and affects transcription up to 1000-fold. The upstream regions is able to retain partial function when positioned within 100-160 nucleotides of the transcription initiation site, but it cannot stimulate transcription from distances of ≥ 600 nucleotides. In addition, they demonstrate, using mouse-human hybrid rRNA promoters, that the sequences responsible for human species-specific transcription in vivo appear to reside in both the core and upstream elements, and sequences from the mouse rRNA promoter cannot be substituted for them

  6. Detection, Characterization, and In Vitro and In Vivo Expression of Genes Encoding S-Proteins in Lactobacillus gallinarum Strains Isolated from Chicken Crops

    Science.gov (United States)

    Hagen, Karen E.; Guan, Le Luo; Tannock, Gerald W.; Korver, Doug R.; Allison, Gwen E.

    2005-01-01

    Thirty-eight isolates of Lactobacillus gallinarum cultured from the crops of broiler chickens were screened for the presence of genes encoding S-layer proteins. All of the isolates had two S-protein genes, which were designated Lactobacillus gallinarum S-protein (lgs) genes. One gene in each isolate was either lgsA or lgsB. The Lactobacillus isolates were further characterized by pulsed-field gel electrophoresis of DNA digests, which grouped the isolates into 17 genotypes (strains). The second gene in each of eight representative strains was sequenced and shown to differ among strains (lgsC, lgsD, lgsE, lgsF, lgsG, lgsH, and lgsI). The genome of each strain thus encoded a common S-protein (encoded by either lgsA or lgsB) and a strain-specific S-protein. The extraction of cell surface proteins from cultures of the eight strains showed that each strain produced a single S-protein that was always encoded by the strain-specific lgs gene. Two of the strains were used to inoculate chickens maintained in a protected environment which were Lactobacillus-free prior to inoculation. DNAs and RNAs extracted from the digesta of the chickens were used for PCR and reverse transcription-PCR, respectively, to demonstrate the presence and transcription of lgs genes in vivo. In both cases, only the strain-specific gene was transcribed. Both of the strains adhered to the crop epithelium, consistent with published data predicting that S-proteins of lactobacilli are adhesins. The results of this study provide a basis for the investigation of gene duplication and sequence variation as mechanisms by which bacterial strains of the same species can share the same habitat. PMID:16269691

  7. Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases

    Science.gov (United States)

    Sauter, Bernhard V.; Martinet, Olivier; Zhang, Wei-Jian; Mandeli, John; Woo, Savio L. C.

    2000-04-01

    Inhibition of angiogenesis has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in the large-scale production of the antiangiogenic proteins. This limitation may be resolved by in vivo delivery and expression of the antiangiogenic genes. We have constructed a recombinant adenovirus that expresses murine endostatin that is biologically active both in vitro, as determined in endothelial cell proliferation assays, and in vivo, by suppression of angiogenesis induced by vascular endothelial growth factor 165. Persistent high serum levels of endostatin (605-1740 ng/ml; mean, 936 ng/ml) were achieved after systemic administration of the vector to nude mice, which resulted in significant reduction of the growth rates and the volumes of JC breast carcinoma and Lewis lung carcinoma (P < 0.001 and P < 0.05, respectively). In addition, the endostatin vector treatment completely prevented the formation of pulmonary micrometastases in Lewis lung carcinoma (P = 0.0001). Immunohistochemical staining of the tumors demonstrated a decreased number of blood vessels in the treatment group versus the controls. In conclusion, the present study clearly demonstrates the potential of vector-mediated antiangiogenic gene therapy as a component in cancer therapy.

  8. In vivo immunologic selection of class I major histocompatibility complex gene deletion variants from the B16-BL6 melanoma.

    Science.gov (United States)

    Talmadge, J E; Talmadge, C B; Zbar, B; McEwen, R; Meeker, A K; Tribble, H

    1987-06-01

    The mechanism by which tumor allografts escape host immunologic attack was investigated. B16-BL6 cells (the bladder 6 subline of the B16 melanoma) (H-2b) were transfected with a gene (Dd) encoding an allogeneic class I major histocompatibility complex antigen. Clones that expressed Dd antigen were injected into the footpads of nonimmune syngeneic mice, syngeneic immune mice, and nude mice. Under conditions of immunologic selection a clone that contained multiple copies of the transfected gene formed variants that lacked the transfected gene. Primary tumors and pulmonary metastases of immunized mice and pulmonary metastases of nonimmunized mice had lost the Dd gene and, in most cases, all of the associated plasmid. In contrast, in immunodeficient nude mice, primary tumors and pulmonary metastases retained the Dd gene and the associated plasmid. Deletion of genes encoding cell surface antigens may be one of the mechanisms by which allogeneic tumors escape immunologic attack.

  9. Doxorubicin in vivo rapidly alters expression and translation of myocardial electron transport chain genes, leads to ATP loss and caspase 3 activation.

    Directory of Open Access Journals (Sweden)

    Amy V Pointon

    2010-09-01

    Full Text Available Doxorubicin is one of the most effective anti-cancer drugs but its use is limited by cumulative cardiotoxicity that restricts lifetime dose. Redox damage is one of the most accepted mechanisms of toxicity, but not fully substantiated. Moreover doxorubicin is not an efficient redox cycling compound due to its low redox potential. Here we used genomic and chemical systems approaches in vivo to investigate the mechanisms of doxorubicin cardiotoxicity, and specifically test the hypothesis of redox cycling mediated cardiotoxicity.Mice were treated with an acute dose of either doxorubicin (DOX (15 mg/kg or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ (25 mg/kg. DMNQ is a more efficient redox cycling agent than DOX but unlike DOX has limited ability to inhibit gene transcription and DNA replication. This allowed specific testing of the redox hypothesis for cardiotoxicity. An acute dose was used to avoid pathophysiological effects in the genomic analysis. However similar data were obtained with a chronic model, but are not specifically presented. All data are deposited in the Gene Expression Omnibus (GEO. Pathway and biochemical analysis of cardiac global gene transcription and mRNA translation data derived at time points from 5 min after an acute exposure in vivo showed a pronounced effect on electron transport chain activity. This led to loss of ATP, increased AMPK expression, mitochondrial genome amplification and activation of caspase 3. No data gathered with either compound indicated general redox damage, though site specific redox damage in mitochondria cannot be entirely discounted.These data indicate the major mechanism of doxorubicin cardiotoxicity is via damage or inhibition of the electron transport chain and not general redox stress. There is a rapid response at transcriptional and translational level of many of the genes coding for proteins of the electron transport chain complexes. Still though ATP loss occurs with activation caspase 3 and these

  10. Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody.

    Science.gov (United States)

    Dhungel, Bidur; Ohno, Yoshikazu; Matayoshi, Rie; Otaki, Joji M

    2013-03-25

    Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally

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

    Directory of Open Access Journals (Sweden)

    Tomoko Nakanishi

    2013-01-01

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

  12. Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.

    Science.gov (United States)

    Coleman, John W; Wright, Kevin J; Wallace, Olivia L; Sharma, Palka; Arendt, Heather; Martinez, Jennifer; DeStefano, Joanne; Zamb, Timothy P; Zhang, Xinsheng; Parks, Christopher L

    2015-03-01

    Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. In vivo preclinical low field MRI monitoring of tumor growth following a suicide gene therapy in an ortho-topic mice model of human glioblastoma

    International Nuclear Information System (INIS)

    Breton, E.; Goetz, Ch.; Aubertin, G.; Constantinesco, A.; Choquet, Ph.; Kintz, J.; Accart, N.; Grellier, B.; Erbs, Ph.; Rooke, R.

    2010-01-01

    Purpose The aim of this study was to monitor in vivo with low field MRI growth of a murine ortho-topic glioma model following a suicide gene therapy. Methods The gene therapy consisted in the stereotactic injection in the mice brain of a modified vaccinia virus Ankara (M.V.A.) vector encoding for a suicide gene (FCU1) that transforms a non toxic pro-drug 5-fluoro-cytosine (5-F.C.) to its highly cytotoxic derivatives 5-fluorouracil (5-F.U.) and 5-fluoro-uridine-5 monophosphate (5-F.U.M.P.). Using a warmed-up imaging cell, sequential 3D T1 and T2 0.1T MRI brain examinations were performed on 16 Swiss female nu/nu mice bearing ortho-topic human glioblastoma (U 87-MG cells). The 6-week in vivo MRI follow-up consisted in a weekly measurement of the intracerebral tumor volume leading to a total of 65 examinations. Mice were divided in four groups: sham group (n = 4), sham group treated with 5-F.C. only (n = 4), sham group with injection of M.V.A.-FCU1 vector only (n = 4), therapy group administered with M.V.A.-FCU1 vector and 5-F.C. (n = 4). Measurements of tumor volumes were obtained after manual segmentation of T1- and T2-weighted images. Results Intra-observer and inter-observer tumor volume measurements show no significant differences. No differences were found between T1 and T2 volume tumor doubling times between the three sham groups. A significant statistical difference (p < 0.05) in T1 and T2 volume tumor doubling times between the three sham groups and the animals treated with the intratumoral injection of M.V.A.-FCU1 vector in combination with 2 weeks per os 5-F.C. administration was demonstrated. Conclusion Preclinical low field MRI was able to monitor efficacy of suicide gene therapy in delaying the tumor growth in an in vivo mouse model of ortho-topic glioblastoma. (authors)

  14. Spatio-Temporal Gene Expression Profiling during In Vivo Early Ovarian Folliculogenesis: Integrated Transcriptomic Study and Molecular Signature of Early Follicular Growth.

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    Agnes Bonnet

    Full Text Available The successful achievement of early ovarian folliculogenesis is important for fertility and reproductive life span. This complex biological process requires the appropriate expression of numerous genes at each developmental stage, in each follicular compartment. Relatively little is known at present about the molecular mechanisms that drive this process, and most gene expression studies have been performed in rodents and without considering the different follicular compartments.We used RNA-seq technology to explore the sheep transcriptome during early ovarian follicular development in the two main compartments: oocytes and granulosa cells. We documented the differential expression of 3,015 genes during this phase and described the gene expression dynamic specific to these compartments. We showed that important steps occurred during primary/secondary transition in sheep. We also described the in vivo molecular course of a number of pathways. In oocytes, these pathways documented the chronology of the acquisition of meiotic competence, migration and cellular organization, while in granulosa cells they concerned adhesion, the formation of cytoplasmic projections and steroid synthesis. This study proposes the involvement in this process of several members of the integrin and BMP families. The expression of genes such as Kruppel-like factor 9 (KLF9 and BMP binding endothelial regulator (BMPER was highlighted for the first time during early follicular development, and their proteins were also predicted to be involved in gene regulation. Finally, we selected a data set of 24 biomarkers that enabled the discrimination of early follicular stages and thus offer a molecular signature of early follicular growth. This set of biomarkers includes known genes such as SPO11 meiotic protein covalently bound to DSB (SPO11, bone morphogenetic protein 15 (BMP15 and WEE1 homolog 2 (S. pombe(WEE2 which play critical roles in follicular development but other biomarkers

  15. Comparison of Expression Profiles in Ovarian Epithelium In Vivo and Ovarian Cancer Identifies Novel Candidate Genes Involved in Disease Pathogenesis

    Science.gov (United States)

    Emmanuel, Catherine; Gava, Natalie; Kennedy, Catherine; Balleine, Rosemary L.; Sharma, Raghwa; Wain, Gerard; Brand, Alison; Hogg, Russell; Etemadmoghadam, Dariush; George, Joshy; Birrer, Michael J.; Clarke, Christine L.; Chenevix-Trench, Georgia; Bowtell, David D. L.; Harnett, Paul R.; deFazio, Anna

    2011-01-01

    Molecular events leading to epithelial ovarian cancer are poorly understood but ovulatory hormones and a high number of life-time ovulations with concomitant proliferation, apoptosis, and inflammation, increases risk. We identified genes that are regulated during the estrous cycle in murine ovarian surface epithelium and analysed these profiles to identify genes dysregulated in human ovarian cancer, using publically available datasets. We identified 338 genes that are regulated in murine ovarian surface epithelium during the estrous cycle and dysregulated in ovarian cancer. Six of seven candidates selected for immunohistochemical validation were expressed in serous ovarian cancer, inclusion cysts, ovarian surface epithelium and in fallopian tube epithelium. Most were overexpressed in ovarian cancer compared with ovarian surface epithelium and/or inclusion cysts (EpCAM, EZH2, BIRC5) although BIRC5 and EZH2 were expressed as highly in fallopian tube epithelium as in ovarian cancer. We prioritised the 338 genes for those likely to be important for ovarian cancer development by in silico analyses of copy number aberration and mutation using publically available datasets and identified genes with established roles in ovarian cancer as well as novel genes for which we have evidence for involvement in ovarian cancer. Chromosome segregation emerged as an important process in which genes from our list of 338 were over-represented including two (BUB1, NCAPD2) for which there is evidence of amplification and mutation. NUAK2, upregulated in ovarian surface epithelium in proestrus and predicted to have a driver mutation in ovarian cancer, was examined in a larger cohort of serous ovarian cancer where patients with lower NUAK2 expression had shorter overall survival. In conclusion, defining genes that are activated in normal epithelium in the course of ovulation that are also dysregulated in cancer has identified a number of pathways and novel candidate genes that may contribute

  16. Widespread promoter methylation of synaptic plasticity genes in long-term potentiation in the adult brain in vivo.

    Science.gov (United States)

    Maag, Jesper L V; Kaczorowski, Dominik C; Panja, Debabrata; Peters, Timothy J; Bramham, Clive R; Wibrand, Karin; Dinger, Marcel E

    2017-03-23

    DNA methylation is a key modulator of gene expression in mammalian development and cellular differentiation, including neurons. To date, the role of DNA modifications in long-term potentiation (LTP) has not been explored. To investigate the occurrence of DNA methylation changes in LTP, we undertook the first detailed study to describe the methylation status of all known LTP-associated genes during LTP induction in the dentate gyrus of live rats. Using a methylated DNA immunoprecipitation (MeDIP)-array, together with previously published matched RNA-seq and public histone modification data, we discover widespread changes in methylation status of LTP-genes. We further show that the expression of many LTP-genes is correlated with their methylation status. We show that these correlated genes are enriched for RNA-processing, active histone marks, and specific transcription factors. These data reveal that the synaptic activity-evoked methylation changes correlates with pre-existing activation of the chromatin landscape. Finally, we show that methylation of Brain-derived neurotrophic factor (Bdnf) CpG-islands correlates with isoform switching from transcripts containing exon IV to exon I. Together, these data provide the first evidence of widespread regulation of methylation status in LTP-associated genes.

  17. Expression of mismatch repair gene PMS2 in nasopharyngeal carcinoma and regulation by glycogen synthase kinase-3β in vivo and in vitro.

    Science.gov (United States)

    Fang, Jugao; Lei, Wenbin; Huang, Xiaoming; Li, Pingdong; Chen, Xiaohong; Zhu, Xiaolin; Wen, Weiping; Li, Huabin

    2012-02-01

    To evaluate the expression of mismatch repair gene PMS2 in human nasopharyngeal carcinoma (NPC) tissues and evaluate the effect of glycogen synthase kinase (GSK)-3β on PMS2 production in vivo and in vitro. The expression of PMS2 and inactivated phosphorylated GSK-3β(s9) was examined by immunohistochemical staining in 25 NPC tissues and the relation was determined by correlation analysis. The effect of GSK-3β transfection in CNE-2 cells on PMS2 production as well as cell apoptosis and chemosensitization were evaluated using small interference RNA (siRNA), immunoblotting and flow cytometric analysis in vitro. The expression of inactivated phosphorylated GSK-3β(s9) was found to negative correlated with PMS2 in vivo. And transfected GSK-3β was found to be able to enhance PMS2 production, and increase cell apoptosis in CNE-2 cells in combination with cisplatin administration in vitro. Inactivation of GSK-3β might be important for NPC tumorgenesis through negatively regulating PMS2 production, and enhanced PMS2 production by GSK-3β is beneficial for understanding the NPC tumorgenesis and developing potential strategy for future therapy. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  18. ERalpha and AP-1 interact in vivo with a specific sequence of the F promoter of the human ERalpha gene in osteoblasts.

    Science.gov (United States)

    Lambertini, Elisabetta; Tavanti, Elisa; Torreggiani, Elena; Penolazzi, Letizia; Gambari, Roberto; Piva, Roberta

    2008-07-01

    Estrogen-responsive genes often have an estrogen response element (ERE) positioned next to activator protein-1 (AP-1) binding sites. Considering that the interaction between ERE and AP-1 elements has been described for the modulation of bone-specific genes, we investigated the 17-beta-estradiol responsiveness and the role of these cis-elements present in the F promoter of the human estrogen receptor alpha (ERalpha) gene. The F promoter, containing the sequence analyzed here, is one of the multiple promoters of the human ERalpha gene and is the only active promoter in bone tissue. Through electrophoretic mobility shift (EMSA), chromatin immunoprecipitation (ChIP), and re-ChIP assays, we investigated the binding of ERalpha and four members of the AP-1 family (c-Jun, c-fos, Fra-2, and ATF2) to a region located approximately 800 bp upstream of the transcriptional start site of exon F of the human ERalpha gene in SaOS-2 osteoblast-like cells. Reporter gene assay experiments in combination with DNA binding assays demonstrated that F promoter activity is under the control of upstream cis-acting elements which are recognized by specific combinations of ERalpha, c-Jun, c-fos, and ATF2 homo- and heterodimers. Moreover, ChIP and re-ChIP experiments showed that these nuclear factors bind the F promoter in vivo with a simultaneous occupancy stimulated by 17-beta-estradiol. Taken together, our findings support a model in which ERalpha/AP-1 complexes modulate F promoter activity under conditions of 17-beta-estradiol stimulation. (c) 2008 Wiley-Liss, Inc.

  19. Identification of a novel set of genes reflecting different in vivo invasive patterns of human GBM cells

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    Monticone Massimiliano

    2012-08-01

    Full Text Available Abstract Background Most patients affected by Glioblastoma multiforme (GBM, grade IV glioma experience a recurrence of the disease because of the spreading of tumor cells beyond surgical boundaries. Unveiling mechanisms causing this process is a logic goal to impair the killing capacity of GBM cells by molecular targeting. We noticed that our long-term GBM cultures, established from different patients, may display two categories/types of growth behavior in an orthotopic xenograft model: expansion of the tumor mass and formation of tumor branches/nodules (nodular like, NL-type or highly diffuse single tumor cell infiltration (HD-type. Methods We determined by DNA microarrays the gene expression profiles of three NL-type and three HD-type long-term GBM cultures. Subsequently, individual genes with different expression levels between the two groups were identified using Significance Analysis of Microarrays (SAM. Real time RT-PCR, immunofluorescence and immunoblot analyses, were performed for a selected subgroup of regulated gene products to confirm the results obtained by the expression analysis. Results Here, we report the identification of a set of 34 differentially expressed genes in the two types of GBM cultures. Twenty-three of these genes encode for proteins localized to the plasma membrane and 9 of these for proteins are involved in the process of cell adhesion. Conclusions This study suggests the participation in the diffuse infiltrative/invasive process of GBM cells within the CNS of a novel set of genes coding for membrane-associated proteins, which should be thus susceptible to an inhibition strategy by specific targeting. Massimiliano Monticone and Antonio Daga contributed equally to this work

  20. Identification of a novel set of genes reflecting different in vivo invasive patterns of human GBM cells.

    Science.gov (United States)

    Monticone, Massimiliano; Daga, Antonio; Candiani, Simona; Romeo, Francesco; Mirisola, Valentina; Viaggi, Silvia; Melloni, Ilaria; Pedemonte, Simona; Zona, Gianluigi; Giaretti, Walter; Pfeffer, Ulrich; Castagnola, Patrizio

    2012-08-17

    Most patients affected by Glioblastoma multiforme (GBM, grade IV glioma) experience a recurrence of the disease because of the spreading of tumor cells beyond surgical boundaries. Unveiling mechanisms causing this process is a logic goal to impair the killing capacity of GBM cells by molecular targeting.We noticed that our long-term GBM cultures, established from different patients, may display two categories/types of growth behavior in an orthotopic xenograft model: expansion of the tumor mass and formation of tumor branches/nodules (nodular like, NL-type) or highly diffuse single tumor cell infiltration (HD-type). We determined by DNA microarrays the gene expression profiles of three NL-type and three HD-type long-term GBM cultures. Subsequently, individual genes with different expression levels between the two groups were identified using Significance Analysis of Microarrays (SAM). Real time RT-PCR, immunofluorescence and immunoblot analyses, were performed for a selected subgroup of regulated gene products to confirm the results obtained by the expression analysis. Here, we report the identification of a set of 34 differentially expressed genes in the two types of GBM cultures. Twenty-three of these genes encode for proteins localized to the plasma membrane and 9 of these for proteins are involved in the process of cell adhesion. This study suggests the participation in the diffuse infiltrative/invasive process of GBM cells within the CNS of a novel set of genes coding for membrane-associated proteins, which should be thus susceptible to an inhibition strategy by specific targeting.Massimiliano Monticone and Antonio Daga contributed equally to this work.

  1. Identification of a novel set of genes reflecting different in vivo invasive patterns of human GBM cells

    International Nuclear Information System (INIS)

    Monticone, Massimiliano; Giaretti, Walter; Pfeffer, Ulrich; Daga, Antonio; Candiani, Simona; Romeo, Francesco; Mirisola, Valentina; Viaggi, Silvia; Melloni, Ilaria; Pedemonte, Simona; Zona, Gianluigi

    2012-01-01

    Most patients affected by Glioblastoma multiforme (GBM, grade IV glioma) experience a recurrence of the disease because of the spreading of tumor cells beyond surgical boundaries. Unveiling mechanisms causing this process is a logic goal to impair the killing capacity of GBM cells by molecular targeting. We noticed that our long-term GBM cultures, established from different patients, may display two categories/types of growth behavior in an orthotopic xenograft model: expansion of the tumor mass and formation of tumor branches/nodules (nodular like, NL-type) or highly diffuse single tumor cell infiltration (HD-type). We determined by DNA microarrays the gene expression profiles of three NL-type and three HD-type long-term GBM cultures. Subsequently, individual genes with different expression levels between the two groups were identified using Significance Analysis of Microarrays (SAM). Real time RT-PCR, immunofluorescence and immunoblot analyses, were performed for a selected subgroup of regulated gene products to confirm the results obtained by the expression analysis. Here, we report the identification of a set of 34 differentially expressed genes in the two types of GBM cultures. Twenty-three of these genes encode for proteins localized to the plasma membrane and 9 of these for proteins are involved in the process of cell adhesion. This study suggests the participation in the diffuse infiltrative/invasive process of GBM cells within the CNS of a novel set of genes coding for membrane-associated proteins, which should be thus susceptible to an inhibition strategy by specific targeting. Massimiliano Monticone and Antonio Daga contributed equally to this work

  2. Identification of Gene Transcription Start Sites and Enhancers Responding to Pulmonary Carbon Nanotube Exposure in Vivo

    DEFF Research Database (Denmark)

    Bornholdt, Jette; Saber, Anne Thoustrup; Lilje, Bait

    2017-01-01

    Increased use of nanomaterials in industry, medicine, and consumer products has raised concerns over their toxicity. To ensure safe use of nanomaterials, understanding their biological effects at the molecular level is crucial. In particular, the regulatory mechanisms responsible for the cascade...... of genes activated by nanomaterial exposure are not well-characterized. To this end, we profiled the genome-wide usage of gene transcription start sites and linked active enhancer regions in lungs of C57BL/6 mice 24 h after intratracheal instillation of a single dose of the multiwalled carbon nanotube...

  3. In Vivo Tumor Gene Delivery Using Novel Peptideticles: pH-Responsive and Ligand Targeted Core-Shell Nanoassembly.

    Science.gov (United States)

    Alipour, Mohsen; Majidi, Asia; Molaabasi, Fatemeh; Sheikhnejad, Reza; Hosseinkhani, Saman

    2018-04-30

    Modulating cancer causing genes with nucleic acid based-molecules as cutting-edge approaches need efficient delivery systems to succeed in clinic. Herein, we report design and fabrication of a novel tissue penetrating Peptideticle with charge-structure switching in tumor microenvironment for an effective gene delivery. The comparative in vitro studies indicate that peptideticles identify and bind to tumor endothelial cells and efficiently penetrate into multicellular tumor spheroid. In addition, negatively charged peptideticle at pH 7.4, prevent unwanted interaction while it's sharp charge-structure switching at pH 6.2-6.9 (e.g.in tumor tissue) facilitates malignant cells penetration. More importantly, upon systemic administration into tumor bearing mice, peptideticles effectively localized in tumor tissue and delivered luciferase gene with a 200-fold higher efficiency compared to their non-pH-responsive counterparts. In conclusion, this study presents a robust nanoassembly of safe materials for high efficient tumor gene delivery. This article is protected by copyright. All rights reserved. © 2018 UICC.

  4. Cell-type specific oxytocin gene expression from AAV delivered promoter deletion constructs into the rat supraoptic nucleus in vivo.

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    Raymond L Fields

    Full Text Available The magnocellular neurons (MCNs in the hypothalamus selectively express either oxytocin (OXT or vasopressin (AVP neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON. Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located -216 to -100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs.

  5. In vivo expression of Salmonella enterica serotype Typhi genes in the blood of patients with typhoid fever in Bangladesh.

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    Alaullah Sheikh

    2011-12-01

    Full Text Available Salmonella enterica serotype Typhi is the cause of typhoid fever. It is a human-restricted pathogen, and few data exist on S. Typhi gene expression in humans.We applied an RNA capture and amplification technique, Selective Capture of Transcribed Sequences (SCOTS, and microarray hybridization to identify S. Typhi transcripts expressed in the blood of five humans infected with S. Typhi in Bangladesh. In total, we detected the expression of mRNAs for 2,046 S. Typhi genes (44% of the S. Typhi genome in human blood; expression of 912 genes was detected in all 5 patients, and expression of 1,100 genes was detected in 4 or more patients. Identified transcripts were associated with the virulence-associated PhoP regulon, Salmonella pathogenicity islands, the use of alternative carbon and energy sources, synthesis and transport of iron, thiamine, and biotin, and resistance to antimicrobial peptides and oxidative stress. The most highly represented group were genes currently annotated as encoding proteins designated as hypothetical, unknown, or unclassified. Of the 2,046 detected transcripts, 1,320 (29% of the S. Typhi genome had significantly different levels of detection in human blood compared to in vitro cultures; detection of 141 transcripts was significantly different in all 5 patients, and detection of 331 transcripts varied in at least 4 patients. These mRNAs encode proteins of unknown function, those involved in energy metabolism, transport and binding, cell envelope, cellular processes, and pathogenesis. We confirmed increased expression of a subset of identified mRNAs by quantitative-PCR.We report the first characterization of bacterial transcriptional profiles in the blood of patients with typhoid fever. S. Typhi is an important global pathogen whose restricted host range has greatly inhibited laboratory studies. Our results suggest that S. Typhi uses a largely uncharacterized genetic repertoire to survive within cells and utilize alternate

  6. In vivo gene expression profiling of the entomopathogenic fungus Beauveria bassiana elucidates its infection stratagems in Anopheles mosquito.

    Science.gov (United States)

    Lai, Yiling; Chen, Huan; Wei, Ge; Wang, Guandong; Li, Fang; Wang, Sibao

    2017-08-01

    The use of entomopathogenic fungi to control mosquitoes is a promising tool for reducing vector-borne disease transmission. To better understand infection stratagems of insect pathogenic fungi, we analyzed the global gene expression profiling of Beauveria bassiana at 36, 60, 84 and 108 h after topical infection of Anopheles stephensi adult mosquitoes using RNA sequencing (RNA-Seq). A total of 5,354 differentially expressed genes (DEGs) are identified over the course of fungal infection. When the fungus grows on the mosquito cuticle, up-regulated DEGs include adhesion-related genes involved in cuticle attachment, Pth11-like GPCRs hypothesized to be involved in host recognition, and extracellular enzymes involved in the degradation and penetration of the mosquito cuticle. Once in the mosquito hemocoel, the fungus evades mosquito immune system probably through up-regulating expression of β-1,3-glucan degrading enzymes and chitin synthesis enzymes for remodeling of cell walls. Moreover, six previous unknown SSCP (small secreted cysteine-rich proteins) are significantly up-regulated, which may serve as "effectors" to suppress host defense responses. B. bassiana also induces large amounts of antioxidant genes to mitigate host-generated exogenous oxidative stress. At late stage of infection, B. bassiana activates a broad spectrum of genes including nutrient degrading enzymes, some transporters and metabolism pathway components, to exploit mosquito tissues and hemolymph as a nutrient source for hyphal growth. These findings establish an important framework of knowledge for further comprehensive elucidation of fungal pathogenesis and molecular mechanism of Beauveria-mosquito interactions.

  7. In vivo targeting of ADAM9 gene expression using lentivirus-delivered shRNA suppresses prostate cancer growth by regulating REG4 dependent cell cycle progression.

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    Che-Ming Liu

    Full Text Available Cancer cells respond to stress by activating a variety of survival signaling pathways. A disintegrin and metalloproteinase (ADAM 9 is upregulated during cancer progression and hormone therapy, functioning in part through an increase in reactive oxygen species. Here, we present in vitro and in vivo evidence that therapeutic targeting of ADAM9 gene expression by lentivirus-delivered small hairpin RNA (shRNA significantly inhibited proliferation of human prostate cancer cell lines and blocked tumor growth in a murine model of prostate cancer bone metastasis. Cell cycle studies confirmed an increase in the G1-phase and decrease in the S-phase population of cancer cells under starvation stress conditions, which correlated with elevated intracellular superoxide levels. Microarray data showed significantly decreased levels of regenerating islet-derived family member 4 (REG4 expression in prostate cancer cells with knockdown of ADAM9 gene expression. This REG4 downregulation also resulted in induction of expression of p21(Cip1/WAF1, which negatively regulates cyclin D1 and blocks the G1/S transition. Our data reveal a novel molecular mechanism of ADAM9 in the regulation of prostate cancer cell proliferation, and suggests a combined modality of ADAM9 shRNA gene therapy and cytotoxic agents for hormone refractory and bone metastatic prostate cancer.

  8. Evidence that expression of a mutated p53 gene attenuates apoptotic cell death in human gastric intestinal-type carcinomas in vivo.

    Science.gov (United States)

    Ishida, M; Gomyo, Y; Ohfuji, S; Ikeda, M; Kawasaki, H; Ito, H

    1997-05-01

    To examine in vivo the validity of the results of experiments in vitro, we analyzed the relationship between p53 gene status and apoptotic cell death of human gastric intestinal-type adenocarcinomas. Surgical specimens were classified into two categories: 18 gastric cancers with nuclear p53 protein (A), and 17 gastric cancers without nuclear p53 protein (B). Polymerase chain reaction-single strand conformation polymorphism disclosed a shifted band that corresponded to a mutation in the p53 gene in 13 cases (72%) in category A and 3 cases (18%) in category B, the frequency being significantly higher in the former (P terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The TUNEL index [TI; (the number of TUNEL-positive apoptotic cells/the total number of tumor cells) x 100] was 3.8 +/- 1.4% in category A and 4.9 +/- 1.2% in category B, the value being significantly lower in the former (P gastric cancer, in accordance with the previous in vitro finding that p53 gene mutation provides a possible selective advantage for tumor cell proliferation, and (2) apoptosis is related not only to expression of p53 and the stage of the cell cycle, but also to p53-independent and cell cycle-independent events.

  9. Correlating Anatomy and Function with Gene Expression in Individual Neurons by Combining in Vivo Labeling, Patch Clamp, and Single Cell RNA-seq

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    Carsten K. Pfeffer

    2017-11-01

    Full Text Available The classification of neurons into distinct types is an ongoing effort aimed at revealing and understanding the diversity of the components of the nervous system. Recently available methods allow us to determine the gene expression pattern of individual neurons in the mammalian cerebral cortex to generate powerful categorization schemes. For a thorough understanding of neuronal diversity such genetic categorization schemes need to be combined with traditional classification parameters like position, axonal projection or response properties to sensory stimulation. Here we describe a method to link the gene expression of individual neurons with their position, axonal projection, or sensory response properties. Neurons are labeled in vivo based on their anatomical or functional properties and, using patch clamp pipettes, their RNA individually harvested in vitro for RNAseq. We validate the methodology using multiple established molecularly and anatomically distinct cell populations and explore molecular differences between uncharacterized neurons in mouse visual cortex. Gene expression patterns between L5 neurons projecting to frontal or contralateral cortex are distinct while L2 neurons differing in position, projection, or function are molecularly similar. With this method we can determine the genetic expression pattern of functionally and anatomically identified individual neurons.

  10. In vitro and in vivo gene delivery using chitosan/hyaluronic acid nanoparticles: Influences of molecular mass of hyaluronic acid and lyophilization on transfection efficiency.

    Science.gov (United States)

    Sato, Toshinori; Nakata, Mitsuhiro; Yang, Zhihong; Torizuka, Yu; Kishimoto, Satoko; Ishihara, Masayuki

    2017-08-01

    Lyophilization is an effective method for preserving nonviral gene vectors. To improve the stability and transgene expression of lyophilized plasmid DNA (pDNA) complexes, we coated the surfaces of pDNA/chitosan complexes with hyaluronic acid (HA) of varying molecular masses. The transgene expression of pDNA/chitosan/HA ternary complexes was characterized in vitro and in vivo. pDNA complexes were lyophilized overnight and the resultant products with spongy, porous consistencies were stored at -30, 4 or 25°C for 2 weeks. Rehydrated complexes were characterized using gel retardation assays, aiming to confirm complex formation, measure particle size and evaluate zeta potential, as well as conduct luciferase gene reporter assays. The anti-tumor effects of pDNA ternary complexes were evaluated using suicide gene (pTK) coding thymidine kinase in Huh7-implanted mice. Transfection efficiencies of pDNA/chitosan/HA ternary complexes were dependent on the average molecular masses of HA. The coating of pDNA/chitosan complexes with HA maintained the cellular transfection efficiencies of lyophilized pDNA ternary complexes. Furthermore, intratumoral injection of lyophilized, rehydrated pDNA ternary complexes into tumor-bearing mice showed a significant suppression of tumor growth. The coating of pDNA/chitosan complexes with high-molecular-weight HA augmented the stability and cellular transfection ability of the complexes after lyophilization-rehydration. Copyright © 2017 John Wiley & Sons, Ltd.

  11. Transformation of Lactuca sativa L. with rol C gene results in increased antioxidant potential and enhanced analgesic, anti-inflammatory and antidepressant activities in vivo.

    Science.gov (United States)

    Ismail, Hammad; Dilshad, Erum; Waheed, Mohammad Tahir; Sajid, Moniba; Kayani, Waqas Khan; Mirza, Bushra

    2016-12-01

    Lettuce is an important edible crop which possesses various medicinal properties. In this study Lactuca sativa L. (cv Grand Rapids) was transformed by Agrobacterium-mediated transformation with rol C gene. Transgene integration and expression was confirmed through PCR and semiquantitative RT-PCR. The transformed extracts were evaluated for their in vitro antioxidant and in vivo analgesic, anti-inflammatory and antidepressant activities in rats. The transformed plants showed 53-98 % increase in total phenolic and 45-58 % increase in total flavonoid contents compared with untransformed plants. Results of total reducing power and total antioxidant capacity exhibited 90-118 and 61-75 % increase in transformed plants, respectively. In contrast to control, DPPH, lipid peroxidation and DNA protection assay showed up to 37, 20 and 50 % enhancement in transformed plants, respectively. The extracts showed similar but significant enhancement behavior in hot plate analgesic and carrageenan-induced hind paw edema test. The transformed extracts showed 72.1 and 78.5 % increase for analgesic and anti-inflammatory activities, respectively. The transformants of rol C gene exhibited prominent antidepressant activity with 64-73 % increase compared with untransformed plants. In conclusion, the present work suggests that transformation with rol C gene can be used to generate lettuce with enhanced medicinally important properties, such as antioxidant, analgesic, anti-inflammatory and antidepressant potential.

  12. Stable RNA interference of ErbB-2 gene synergistic with epirubicin suppresses breast cancer growth in vitro and in vivo

    International Nuclear Information System (INIS)

    Hu Xiaoqu; Su Fengxi; Qin Li; Jia Weijuan; Gong Chang; Yu Fengyan; Guo Jujiang; Song Erwei

    2006-01-01

    Overexpression of human epidermal growth factor receptor-2 (Her2, ErbB-2) contributes to the progression and metastasis of breast cancer, implying that Her2 gene is a suitable target of RNA interference (RNAi) for breast cancer therapy. Here, we employed plasmid-mediated expression of 2 different Her2-shRNAs (pU6-Her2shRNAs) efficiently silenced the target gene expression on Her2 expressing SKBR-3 breast cancer cells in both mRNA and protein levels. Consequently, pU6-Her2shRNA increased apoptosis and reduced proliferation of SKBR-3 cells assayed by TUNEL and MTT, respectively. In vivo, intra-tumor injection of pU6-Her2shRNA inhibited the growth of SKBR-3 tumors inoculated subcutaneously in nude mice. Furthermore, pU6-Her2shRNA synergized the tumor suppression effect of epirubicin to SKBR-3 cells in vitro and implanted subcutaneously in nude mice. Therefore, we concluded that stable silencing of Her2 gene expression with plasmid expressing shRNA may hold great promise as a novel therapy for Her2 expressing breast cancers alone or in combination with anthracycline chemotherapy

  13. Influence of the IL-1Ra gene polymorphism on in vivo synthesis of IL-1Ra and IL-1beta after live yellow fever vaccination.

    Science.gov (United States)

    Hacker, U T; Erhardt, S; Tschöp, K; Jelinek, T; Endres, S

    2001-09-01

    The inflammatory response in infectious and autoimmune diseases is regulated by the balance between pro- and anti-inflammatory cytokines. The IL-1 complex contains polymorphic genes coding for IL-1alpha, IL-1beta and IL-1Ra. The IL-1Ra (variable number of tanden repeat) VNTR polymorphism has been shown to influence the capacity to produce IL-1beta and IL-1Ra after in vitro stimulation. Allele 2 of this polymorphism is associated with a number of inflammatory diseases. To determine the impact of the IL-1Ra polymorphism on in vivo human cytokine synthesis, we used a yellow fever vaccination model for the induction of cytokine synthesis in healthy volunteers. Two different yellow fever vaccines were used. After administration of the RKI vaccine (34 volunteers), plasma TNF-alpha concentration increased from 13.4 +/- 0.9 pg/ml to 23.3 +/- 1.1 pg/ml (P < 0.001), and plasma IL-1Ra concentration increased from 308 +/- 25 pg/ml to 1019 +/- 111 pg/ml (P < 0.001), on day 2. Using Stamaril vaccine, no increase in the plasma concentrations of either TNF-alpha or IL-1Ra could be detected (n = 17). Only the RKI vaccine induced TNF-alpha synthesis after in vitro stimulation of MNC. Carriers of allele 2 of the IL-1Ra polymorphism had increased baseline concentrations of IL-1Ra (350 +/- 32 pg/ml) compared with non-carriers (222 +/- 18 pg/ml, P < 0.001), and decreased concentrations of IL-1beta (0.9 +/- 0.2 pg/ml for carriers versus 2.8 +/- 0.7 pg/ml for non-carriers, P = 0.017). After yellow fever vaccination (RKI vaccine), no significant differences in the increase of IL-1Ra plasma levels were detected between carriers and non-carriers of allele 2 of the IL-1Ra gene polymorphism. This is the first study to examine the influence of this genetic polymorphism on in vivo-induced human IL-1beta and IL-1Ra synthesis. Baseline concentrations of IL-1Ra and IL-1beta were significantly influenced by the IL-1Ra polymorphism. No influence of the IL-1Ra polymorphism on the in vivo

  14. NF-kappaB specifically activates BMP-2 gene expression in growth plate chondrocytes in vivo and in a chondrocyte cell line in vitro.

    Science.gov (United States)

    Feng, Jian Q; Xing, Lianping; Zhang, Jiang-Hong; Zhao, Ming; Horn, Diane; Chan, Jeannie; Boyce, Brendan F; Harris, Stephen E; Mundy, Gregory R; Chen, Di

    2003-08-01

    Bone morphogenetic protein-2 (BMP-2) regulates growth plate chondrogenesis during development and postnatal bone growth, but the control mechanisms of BMP-2 expression in growth plate chondrocytes are unknown. Here we have used both in vitro and in vivo approaches to demonstrate that transcription factor, NF-kappaB, regulates BMP-2 gene expression in chondrocytes. Two putative NF-kappaB response elements were found in the -2712/+165 region of the BMP-2 gene. Cotransfection of mutant I-kappaBalpha expression plasmids with BMP-2 promoter-luciferase reporters into TMC-23 chondrocyte cell line suppressed BMP-2 transcription. Mutations in NF-kappaB response elements in the BMP-2 gene lead to decreases in BMP-2 promoter activity. Electrophoretic mobility shift assay using nuclear extracts from TMC-23 chondrocytic cells revealed that the NF-kappaB subunits p50 and p65 bound to the NF-kappaB response elements of the BMP-2 gene. Thus, NF-kappaB may positively regulate BMP-2 gene transcription. Consistent with these findings, expression of BMP-2 mRNA was significantly reduced in growth plate chondrocytes in NF-kappaB p50/p52 dKO mice, which associated with decreased numbers of 5-bromo-2'-deoxyuridine (BrdUrd)-positive cells in the proliferating zone of growth plate in these mice. Therefore, in postnatal growth plate chondrocytes, expression of BMP-2 is regulated by NF-kappaB, which may play an important role in chondrogenesis.

  15. Biological nanoparticles carrying the Hmda-7 gene are effective in inhibiting pancreatic cancer in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Qingyun Zhu

    Full Text Available Pancreatic cancer is one of the most common malignancies of the digestive system, and remains a clinical challenge. This study aimed to assess the effects of bovine serum albumin (BSA nanoparticles carrying the hMDA-7 gene (BSA-NP-hMDA-7 in the treatment of pancreatic cancer.BSA-NP-hMDA-7 was generated by nanotechnology and gene recombination technology. A total of 5 BXPC-3 or PANC-1 pancreatic cancer cell groups were examined, including Control, BSA-NPs, Empty vector, hMDA-7 plasmid, and hMDA-7 BSA-NPs groups, respectively. Proliferation and apoptosis of cultured cells were assessed by the MTT method and flow-cytometry, respectively. In addition, pancreatic cancer models were established with both cell lines in nude mice, and the expression profiles of hMDA-7 and VEGF in cancer tissues were measured by Western blot and immunohistochemistry.BSA-NP-hMDA-7 nanoparticles were successfully generated, and significantly inhibited the proliferation of BXPC-3 and PANC-1 cells; in addition, apoptosis rates were higher in both cell lines after treatment with BSA-NP-hMDA-7 (P<0.05. Nude mouse xenograft studies indicated that treatment with BSA-NP-hMDA-7 nanoparticles resulted in decreased tumor size. Moreover, the hMDA-7 protein was found in tumor tissues after hMDA-7 gene transfection, while BSA-NP-hMDA-7 significantly suppressed VEGF expression in tumor tissues. Similar results were obtained for both BXPC-3 and PANC-1 xenograft models.BSA nanoparticles carrying the hMDA-7 gene effectively transfected BXPC-3 and PANC-1 pancreatic cancer cells, causing reduced cell proliferation and enhanced apoptosis in vitro. In mouse xenografts, BSA-NP-hMDA-7 treatment decreased tumor size and reduced VEGF expression. These findings indicated that BSA-NP-hMDA-7 might exert anticancer effects via VEGF suppression.

  16. Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo.

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    Sebastian Frese

    Full Text Available Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human

  17. Effects of in vivo exposure to UV filters (4-MBC, OMC, BP-3, 4-HB, OC, OD-PABA) on endocrine signaling genes in the insect Chironomus riparius.

    Science.gov (United States)

    Ozáez, Irene; Martínez-Guitarte, José Luis; Morcillo, Gloria

    2013-07-01

    There is increasing evidence indicating that several UV filters might have endocrine disruptive effects. Numerous studies have evaluated hormonal effects in vertebrates, mainly reporting estrogenic and androgenic activities in mammals and fishes. There is only limited knowledge about potential endocrine activity in invertebrate hormonal systems. In this work, the effects on endocrine signaling genes of six frequently used UV filters were investigated in Chironomus riparius, a reference organism in aquatic toxicology. The UV filters studied were: octyl-p-methoxycinnamate (OMC) also called 2-ethylhexyl-4-methoxycinnamate (EHMC); 4-methylbenzylidene camphor (4-MBC); benzophenone-3 (BP-3); 4-hidroxybenzophenone (4-HB); octocrylene (OC); and octyldimethyl-p-aminobenzoate (OD-PABA). After in vivo exposure at different dosages, expression levels of the genes coding for the ecdysone receptor (EcR), the ultraspiracle (usp, ortholog of the RXR) and the estrogen-related receptor (ERR) were quantified by Real Time PCR. The EcR gene was significantly upregulated by 4-MBC, OMC/EHMC and OD-PABA, with a dose-related response following 24h exposure. In contrast, the benzophenones, BP-3 and 4-HB, as well as OC did not alter this gene at the same exposure conditions. The transcription profiles of the usp and ERR genes were not significantly affected, except for BP-3 that inhibited the usp gene at the highest concentration. To our knowledge, this is the first experimental evidence in invertebrates of a direct effect of UV filters on endocrine-related genes, and is consistent with the known effects on vertebrate hormonal receptor genes. The capability of 4-MBC, OMC/EHMC and OD-PABA to stimulate the expression of the ecdysone receptor, a key transcription factor for the ecdysone-genomic response in arthropods, suggests the possibility of a broad and long-term effect on this hormonal pathway. These findings strengthen the need for further research about the ecotoxicological implications

  18. Targeted retrograde transfection of adenovirus vector carrying brain-derived neurotrophic factor gene prevents loss of mouse (twy/twy) anterior horn neurons in vivo sustaining mechanical compression.

    Science.gov (United States)

    Xu, Kan; Uchida, Kenzo; Nakajima, Hideaki; Kobayashi, Shigeru; Baba, Hisatoshi

    2006-08-01

    Immunohistochemical analysis after adenovirus (AdV)-mediated BDNF gene transfer in and around the area of mechanical compression in the cervical spinal cord of the hyperostotic mouse (twy/twy). To investigate the neuroprotective effect of targeted AdV-BDNF gene transfection in the twy mouse with spontaneous chronic compression of the spinal cord motoneurons. Several studies reported the neuroprotective effects of neurotrophins on injured spinal cord. However, no report has described the effect of targeted retrograde neurotrophic gene delivery on motoneuron survival in chronic compression lesions of the cervical spinal cord resembling lesions of myelopathy. LacZ marker gene using adenoviral vector (AdV-LacZ) was used to evaluate retrograde delivery from the sternomastoid muscle in adult twy mice (16-week-old) and (control). Four weeks after the AdV-LacZ or AdV-BDNF injection, the compressed cervical spinal cord was removed en bloc for immunohistologic investigation of b-galactosidase activity and immunoreactivity and immunoblot analyses of BDNF. The number of anterior horn neurons was counted using Nissl, ChAT and AChE staining. Spinal accessory motoneurons between C1 and C3 segments were successfully transfected by AdV-LacZ in both twy and ICR mice after targeted intramuscular injection. Immunoreactivity to BDNF was significantly stronger in AdV-BDNF-gene transfected twy mice than in AdV-LacZ-gene transfected mice. At the cord level showing the maximum compression in AdV-BDNF-transfected twy mice, the number of anterior horn neurons was sinificantly higher in the topographic neuronal cell counting of Nissl-, ChAT-, and AChE-stained samples than in AdV-LacZ-injected twy mice. Targeted AdV-BDNF-gene delivery significantly increased Nissl-stained anterior horn neurons and enhanced cholinergic enzyme activities in the twy. Our results suggest that targeted retrograde AdV-BDNF-gene in vivo delivery may enhance neuronal survival even under chronic mechanical compression.

  19. In vivo expression of the lacY gene in two segments leads to functional lac permease

    International Nuclear Information System (INIS)

    Bibi, E.; Kaback, H.R.

    1990-01-01

    The lacY gene of Escherichia coli was cut into two approximately equal-size fragments with Afl II and subcloned individually or together under separate lac operator/promoters in plasmid pT7-5. Under these conditions, lac permease is expressed in two portions: (i) the N-terminal portion (the N terminus, the first six putative transmembrane helices, and most of putative loop 7) and (ii) the C-terminal portion (the last six putative transmembrane helices and the C terminus). Cells harboring pT7-5 encoding both fragments transport lactose at about 30% the rate of cells expressing intact permease to a comparable steady-state level of accumulation. In contrast, cells expressing either half of the permease independently do not transport lactose. As judged by [ 35 S]methionine labeling and immunoblotting, intact permease in completely absent from the membrane of cells expressing lacY fragments either individually or together. Thus, transport activity must result from an association between independently synthesized pieces of lac permease. When the gene fragments are expressed individually, the N-terminal portion of the permease is observed inconsistently, and the C-terminal portion is not observed. When the gene fragments are expressed together, polypeptides identified as the N- and C-terminal moieties of the permease are found in the membrane. It is concluded that the N- or C-terminal halves of lac permease are proteolyzed when synthesized independently and that association between the two complementing polypeptides leads to a more stable, catalytically active complex

  20. The effect of endocrine disruptors on reproductive parameters and expression of selected testicular genes in male mice in vivo

    Czech Academy of Sciences Publication Activity Database

    Pěknicová, Jana; Elzeinová, Fatima; Žatecká, Eva; Děd, Lukáš; Dorosh, Andriy

    2012-01-01

    Roč. 67, Issue Supplement s1 (2012), s. 20 ISSN 1046-7408. [13th International Symposium for Immunology of reproduction "From the roots to the tops of Reproductive Immunology". 22.06.2012-24.06.2012, Varna] R&D Projects: GA ČR(CZ) GA523/09/1793; GA ČR(CZ) GAP503/12/1834 Institutional research plan: CEZ:AV0Z50520701 Keywords : endocrine disruptors * testicular gene * reproductive parameters * reproduction Subject RIV: DN - Health Impact of the Environment Quality

  1. In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine-based nanoparticles

    Directory of Open Access Journals (Sweden)

    Lopes CDF

    2016-06-01

    Full Text Available Cátia DF Lopes,1–3,* Hugo Oliveira,1,* Inês Estevão,1 Liliana Raquel Pires,1 Ana Paula Pêgo1,2,4,5 1INEB – Instituto de Engenharia Biomédica, Universidade do Porto (UPorto, Porto, Portugal; 2i3S – Instituto de Investigação e Inovação em Saúde, NanoBiomaterials for Targeted Therapies Group, UPorto, Porto, Portugal; 3FMUP – Faculdade de Medicina da Universidade do Porto, Porto, Portugal; 4ICBAS – Instituto de Ciências Biomédicas Abel Salazar, UPorto, Porto, Portugal; 5FEUP – Faculdade de Engenharia da Universidade do Porto, Porto, Portugal *These authors contributed equally to this work Abstract: A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC, which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral

  2. Selection of reference genes in different myocardial regions of an in vivo ischemia/reperfusion rat model for normalization of antioxidant gene expression

    Directory of Open Access Journals (Sweden)

    Vesentini Nicoletta

    2012-02-01

    Full Text Available Abstract Background Changes in cardiac gene expression due to myocardial injury are usually assessed in whole heart tissue. However, as the heart is a heterogeneous system, spatial and temporal heterogeneity is expected in gene expression. Results In an ischemia/reperfusion (I/R rat model we evaluated gene expression of mitochondrial and cytoplasmatic superoxide dismutase (MnSod, Cu-ZnSod and thioredoxin reductase (trxr1 upon short (4 h and long (72 h reperfusion times in the right ventricle (RV, and in the ischemic/reperfused (IRR and the remote region (RR of the left ventricle. Gene expression was assessed by Real-time reverse-transcription quantitative PCR (RT-qPCR. In order to select most stable reference genes suitable for normalization purposes, in each myocardial region we tested nine putative reference genes by geNorm analysis. The genes investigated were: Actin beta (actb, Glyceraldehyde-3-P-dehydrogenase (gapdh, Ribosomal protein L13A (rpl13a, Tyrosine 3-monooxygenase (ywhaz, Beta-glucuronidase (gusb, Hypoxanthine guanine Phosphoribosyltransferase 1 (hprt, TATA binding box protein (tbp, Hydroxymethylbilane synthase (hmbs, Polyadenylate-binding protein 1 (papbn1. According to our findings, most stable reference genes in the RV and RR were hmbs/hprt and hmbs/tbp/hprt respectively. In the IRR, six reference genes were recommended for normalization purposes; however, in view of experimental feasibility limitations, target gene expression could be normalized against the three most stable reference genes (ywhaz/pabp/hmbs without loss of sensitivity. In all cases MnSod and Cu-ZnSod expression decreased upon long reperfusion, the former in all myocardial regions and the latter in IRR alone. trxr1 expression did not vary. Conclusions This study provides a validation of reference genes in the RV and in the anterior and posterior wall of the LV of cardiac ischemia/reperfusion model and shows that gene expression should be assessed separately in

  3. Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

    International Nuclear Information System (INIS)

    Zuo, Keqiang; Li, Dan; Pulli, Benjamin; Yu, Fei; Cai, Haidong; Yuan, Xueyu; Zhang, Xiaoping; Lv, Zhongwei

    2012-01-01

    Highlights: ► Hsp90 is over-expressed in human breast cancer. ► The shRNA-mediated gene silencing of Hsp90 resulted in inhibition of cell growth. ► Akt and NF-kB were down-regulation after transfection due to Hsp90 silencing. ► The tumor growth ratio was decline due to Hsp90 silencing. ► The PCNA expression was down-regulation due to Hsp90 silencing. -- Abstract: Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.

  4. Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

    Science.gov (United States)

    Kułdo, J M; Ásgeirsdóttir, S A; Zwiers, P J; Bellu, A R; Rots, M G; Schalk, J A C; Ogawara, K I; Trautwein, C; Banas, B; Haisma, H J; Molema, G; Kamps, J A A M

    2013-02-28

    In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Mouse Mammary Tumor Virus Promoter-Containing Retroviral Promoter Conversion Vectors for Gene-Directed Enzyme Prodrug Therapy are Functional in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Reinhard Klein

    2008-01-01

    Full Text Available Gene directed-enzyme prodrug therapy (GDEPT is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1 metabolizes the prodrugs cyclophosphamide (CPA and ifosfamide (IFA to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC50 values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.

  6. Effects of calcitonin gene-related peptide on canine cerebral artery strips and the in-vivo vertebral blood flow in dogs.

    Science.gov (United States)

    Ikegaki, I; Suzuki, Y; Satoh, S; Asano, T; Shibuya, M; Sugita, K

    1989-10-01

    The effects of calcitonin gene-related peptide (CGRP) on canine cerebral arteries and on vertebral blood flow were investigated in-vivo and in-vitro and the findings compared with the effects of vasoactive intestinal peptide (VIP) and substance P. Administration of CGRP into the vertebral artery caused a dose-dependent and long-lasting increase in blood flow. The in-vivo vasodilatory effects of substance P and VIP were short-lasting. CGRP (0.1 to 100 nmol/l) elicited a concentration-dependent relaxation of the isolated middle cerebral and basilar arteries when the tissues were precontracted by exposure to prostaglandin F2 alpha (PGF2 alpha). This effect was not antagonized by propranolol, atropine, tetrodotoxin, (N-Ac-Tyr1, D-Phe2)-growth hormone-releasing factor(1-29)-NH2 or (D-Pro2, D-Trp7,9) substance P. CGRP also reduced concentration-dependently the contraction of cerebral arteries induced by KCl or 9,11-epithio-11,12-metano-thromboxane A2 (STXA2). Mechanical removal of the endothelium did not abolish the vasodilatory response to CGRP. In PGF2 alpha-contracted canine cerebral arteries, VIP (0.1 to 100 nmol/l) was less potent a vasodilator than CGRP. At low concentrations (0.01 to 1 nmol/l) substance P elicited a rapid and short-lasting relaxation, and in the absence of endothelium this relaxation disappeared. These findings are clear evidence that CGRP modulates vascular tone.

  7. Long and noncoding RNAs (lnc-RNAs determine androgen receptor dependent gene expression in prostate cancer growth in vivo

    Directory of Open Access Journals (Sweden)

    Richard G Pestell

    2014-04-01

    Full Text Available Hyperactive androgen receptor (AR activity remains a key determinant of the onset and progression of prostate cancer and resistance to current therapies. The mechanisms governing castrate resistant prostate cancer are poorly understood, but defining these molecular events is essential in order to impact deaths from prostate cancer. Yang et al. demonstrate that two lnc-RNAs known to be overexpressed in therapy resistant prostate cancer, PRNCR1 (also known as PCAT8 and PCGEM1, bound to the AR to enhance ligand-dependent and ligand-independent AR gene expression and proliferation of prostate cancer cells.1 The sequence of these interactions involved the binding of PRNCR1 to the acetylated AR and a subsequent association of DOT1L, which was required for the sequential recruitment of the lncRNA PCGEM1 to the AR amino terminus, which in turn was methylated by DOT1L.

  8. Suppression of human papillomavirus gene expression in vitro and in vivo by herpes simplex virus type 2 infection

    International Nuclear Information System (INIS)

    Fang, L.; Ward, M.G.; Welsh, P.A.; Budgeon, L.R.; Neely, E.B.; Howett, M.K.

    2003-01-01

    Recent epidemiological studies have found that women infected with both herpes simplex virus type 2 (HSV-2) and human papillomavirus (HPV) type 16 or HPV-18 are at greater risk of developing cervical carcinoma compared to women infected with only one virus. However, it remains unclear if HSV-2 is a cofactor for cervical cancer or if HPV and HSV-2 interact in any way. We have studied the effect of HSV-2 infection on HPV-11 gene expression in an in vitro double-infection assay. HPV transcripts were down-regulated in response to HSV-2 infection. Two HSV-2 vhs mutants failed to reduce HPV-16 E1-circumflexE4 transcripts. We also studied the effect of HSV-2 infection on preexisting experimental papillomas in a vaginal epithelial xenograft model. Doubly infected grafts demonstrated papillomatous transformation and the classical cytopathic effect from HSV-2 infection. HPV and HSV DNA signals were mutually exclusive. These studies may have therapeutic applications for HPV infections and related neoplasms

  9. Of the Nine Cytidine Deaminase-Like Genes in Arabidopsis, Eight Are Pseudogenes and Only One Is Required to Maintain Pyrimidine Homeostasis in Vivo.

    Science.gov (United States)

    Chen, Mingjia; Herde, Marco; Witte, Claus-Peter

    2016-06-01

    CYTIDINE DEAMINASE (CDA) catalyzes the deamination of cytidine to uridine and ammonia in the catabolic route of C nucleotides. The Arabidopsis (Arabidopsis thaliana) CDA gene family comprises nine members, one of which (AtCDA) was shown previously in vitro to encode an active CDA. A possible role in C-to-U RNA editing or in antiviral defense has been discussed for other members. A comprehensive bioinformatic analysis of plant CDA sequences, combined with biochemical functionality tests, strongly suggests that all Arabidopsis CDA family members except AtCDA are pseudogenes and that most plants only require a single CDA gene. Soybean (Glycine max) possesses three CDA genes, but only two encode functional enzymes and just one has very high catalytic efficiency. AtCDA and soybean CDAs are located in the cytosol. The functionality of AtCDA in vivo was demonstrated with loss-of-function mutants accumulating high amounts of cytidine but also CMP, cytosine, and some uridine in seeds. Cytidine hydrolysis in cda mutants is likely caused by NUCLEOSIDE HYDROLASE1 (NSH1) because cytosine accumulation is strongly reduced in a cda nsh1 double mutant. Altered responses of the cda mutants to fluorocytidine and fluorouridine indicate that a dual specific nucleoside kinase is involved in cytidine as well as uridine salvage. CDA mutants display a reduction in rosette size and have fewer leaves compared with the wild type, which is probably not caused by defective pyrimidine catabolism but by the accumulation of pyrimidine catabolism intermediates reaching toxic concentrations. © 2016 American Society of Plant Biologists. All Rights Reserved.

  10. Expression patterns of the hypoxia-related genes osteopontin, CA9, erythropoietin, VEGF and HIF-1α in human glioma in vitro and in vivo

    International Nuclear Information System (INIS)

    Said, Harun M.; Hagemann, Carsten; Staab, Adrian; Stojic, Jelena; Kuehnel, Siglinde; Vince, Giles H.; Flentje, Michael; Roosen, Klaus; Vordermark, Dirk

    2007-01-01

    Background and purpose: To identify molecular markers of tumor hypoxia and potential therapeutic targets in glioblastoma (GBM), we investigated the hypoxia-related expression of osteopontin (OPN), carbonic anhydrase 9 (CA9), erythropoietin (EPO), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) in vitro in human GBM cell lines and in vivo in human tumor samples of GBM, compared to low-grade astrocytoma (LGA). Materials and methods: Expression of the hypoxia-induced genes OPN, CA9, EPO, VEGF and HIF-1α was analyzed in three GBM cell lines, GaMG, U373 and U251, under in vitro hypoxia (1, 6 or 24 h at 5%, 1% or 0.1% O 2 ) and in tumor samples from two patient groups with LGA and GBM (n = 15 each), at the mRNA level (semiquantitative RT-PCR). Selected conditions and representative tumor samples were also evaluated at the protein level by Western blot. Results: OPN and CA9 mRNA was most consistently upregulated in relation to severity and duration of in vitro hypoxia. In tumor samples, mean expression levels (LGA vs. GBM, normalized to mean expression in normal brain) were 1.71 vs. 4.57 (p < 0.001) for OPN, 1.11 vs. 3.35 (p < 0.001) for CA9, 2.79 vs. 5.28 (not significant, n.s.) for Epo, 1.13 vs. 2.0 (p = 0.007) for VEGF and 0.97 vs. 0.97 (n.s.) for HIF-1α. In tumor samples, GBM showed a particularly strong protein expression of OPN. Conclusions: Among a panel of known hypoxia-inducible genes, OPN and CA9 emerge as most consistently induced by in vitro hypoxia in human GBM cell lines and most specifically expressed in patient GBM tumor tissue, rendering these two genes attractive targets for hypoxia-directed treatment approaches

  11. Involvement of Histone Lysine Methylation in p21 Gene Expression in Rat Kidney In Vivo and Rat Mesangial Cells In Vitro under Diabetic Conditions

    Directory of Open Access Journals (Sweden)

    Xiangjun Li

    2016-01-01

    Full Text Available Diabetic nephropathy (DN, a common complication associated with type 1 and type 2 diabetes mellitus (DM, characterized by glomerular mesangial expansion, inflammation, accumulation of extracellular matrix (ECM protein, and hypertrophy, is the major cause of end-stage renal disease (ESRD. Increasing evidence suggested that p21-dependent glomerular and mesangial cell (MC hypertrophy play key roles in the pathogenesis of DN. Recently, posttranscriptional modifications (PTMs have uncovered novel molecular mechanisms involved in DN. However, precise regulatory mechanism of histone lysine methylation (HKme mediating p21 related hypertrophy associated with DN is not clear. We evaluated the roles of HKme and histone methyltransferase (HMT SET7/9 in p21 gene expression in glomeruli of diabetic rats and in high glucose- (HG- treated rat mesangial cells (RMCs. p21 gene expression was upregulated in diabetic rats glomeruli; chromatin immunoprecipitation (ChIP assays showed decreased histone H3-lysine9-dimethylation (H3K9me2 accompanied with enhanced histone H3-lysine4-methylation (H3K4me1/3 and SET7/9 occupancies at the p21 promoter. HG-treated RMCs exhibited increased p21 mRNA, H3K4me level, SET7/9 recruitment, and inverse H3K9me, which were reversed by TGF-β1 antibody. These data uncovered key roles of H3Kme and SET7/9 responsible for p21 gene expression in vivo and in vitro under diabetic conditions and confirmed preventive effect of TGF-β1 antibody on DN.

  12. SpxB is a suicide gene of Streptococcus pneumoniae and confers a selective advantage in an in vivo competitive colonization model.

    Science.gov (United States)

    Regev-Yochay, Gili; Trzcinski, Krzysztof; Thompson, Claudette M; Lipsitch, Marc; Malley, Richard

    2007-09-01

    The human bacterial pathogen Streptococcus pneumoniae dies spontaneously upon reaching stationary phase. The extent of S. pneumoniae death at stationary phase is unusual in bacteria and has been conventionally attributed to autolysis by the LytA amidase. In this study, we show that spontaneous pneumococcal death is due to hydrogen peroxide (H(2)O(2)), not LytA, and that the gene responsible for H(2)O(2) production (spxB) also confers a survival advantage in colonization. Survival of S. pneumoniae in stationary phase was significantly prolonged by eliminating H(2)O(2) in any of three ways: chemically by supplementing the media with catalase, metabolically by growing the bacteria under anaerobic conditions, or genetically by constructing DeltaspxB mutants that do not produce H(2)O(2). Likewise, addition of H(2)O(2) to exponentially growing S. pneumoniae resulted in a death rate similar to that of cells in stationary phase. While DeltalytA mutants did not lyse at stationary phase, they died at a rate similar to that of the wild-type strain. Furthermore, we show that the death process induced by H(2)O(2) has features of apoptosis, as evidenced by increased annexin V staining, decreased DNA content, and appearance as assessed by transmission electron microscopy. Finally, in an in vivo rat model of competitive colonization, the presence of spxB conferred a selective advantage over the DeltaspxB mutant, suggesting an explanation for the persistence of this gene. We conclude that a suicide gene of pneumococcus is spxB, which induces an apoptosis-like death in pneumococci and confers a selective advantage in nasopharyngeal cocolonization.

  13. Partial rescue of in vivo insulin signalling in skeletal muscle by impaired insulin clearance in heterozygous carriers of a mutation in the insulin receptor gene

    DEFF Research Database (Denmark)

    Højlund, K.; Wojtaszewski, Jørgen; Birk, Jesper Bratz

    2006-01-01

    AIMS/HYPOTHESIS: Recently we reported the coexistence of postprandial hypoglycaemia and moderate insulin resistance in heterozygous carriers of the Arg1174Gln mutation in the insulin receptor gene (INSR). Controlled studies of in vivo insulin signalling in humans with mutant INSR are unavailable,...

  14. Radiosensitization and growth inhibition of cancer cells mediated by an scFv antibody gene against DNA-PKcs in vitro and in vivo

    International Nuclear Information System (INIS)

    Du, Li; Zhou, Ping-Kun; Zhou, Li-Jun; Pan, Xiu-Jie; Wang, Yu-Xiao; Xu, Qin-Zhi; Yang, Zhi-Hua; Wang, Yu; Liu, Xiao-Dan; Zhu, Mao-Xiang

    2010-01-01

    Overexpression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is commonly occurred in cancers and causes radioresistance and poor prognosis. In present study, the single-chain variable antibody fragments (scFv) targeting DNA-PKcs was developed for the application of radiosensitization in vitro and in vivo. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. DNA-PKcs epitopes were predicted and cloned. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. DNA damage repair was analyzed by comet assay and immunofluorescence detection of γH2AX foci. The radiosensitization in vivo was determined on Balb/c athymic mice transplanted tumours of HeLa cells. Four epitopes of DNA-PKcs have been predicted and expressed as the antigens, and a specific human anti-DNA-PKcs scFv antibody gene, anti-DPK3-scFv, was obtained by screening the phage antibody library using the DNA-PKcs peptide DPK3. The specificity of anti-DPK3-scFv was verified, in vitro. Transfection of HeLa cells with the anti-DPK3-scFv gene resulted in an increased sensitivity to IR, decreased repair capability of DNA double-strand breaks (DSB) detected by comet assay and immunofluorescence detection of γH2AX foci. Moreover, the kinase activity of DNA-PKcs was inhibited by anti-DPK3-scFv, which was displayed by the decreased phosphorylation levels of its target Akt/S473 and the autophosphorylation of DNA-PKcs on S2056 induced by radiation. Measurement of the growth and apoptosis rates showed that anti-DPK3-scFv enhanced the sensitivity of tumours transplanted in Balb/c athymic mice to radiation therapy. The antiproliferation and radiosensitizing effects of anti-DPK3-scFv via targeting DNA-PKcs make it very appealing for the development as a novel biological radiosensitizer for cancer therapeutic potential

  15. A laser pointer driven microheater for precise local heating and conditional gene regulation in vivo. Microheater driven gene regulation in zebrafish

    Directory of Open Access Journals (Sweden)

    Achermann Marc

    2009-12-01

    Full Text Available Abstract Background Tissue heating has been employed to study a variety of biological processes, including the study of genes that control embryonic development. Conditional regulation of gene expression is a particularly powerful approach for understanding gene function. One popular method for mis-expressing a gene of interest employs heat-inducible heat shock protein (hsp promoters. Global heat shock of hsp-promoter-containing transgenic animals induces gene expression throughout all tissues, but does not allow for spatial control. Local heating allows for spatial control of hsp-promoter-driven transgenes, but methods for local heating are cumbersome and variably effective. Results We describe a simple, highly controllable, and versatile apparatus for heating biological tissue and other materials on the micron-scale. This microheater employs micron-scale fiber optics and uses an inexpensive laser-pointer as a power source. Optical fibers can be pulled on a standard electrode puller to produce tips of varying sizes that can then be used to reliably heat 20-100 μm targets. We demonstrate precise spatiotemporal control of hsp70l:GFP transgene expression in a variety of tissue types in zebrafish embryos and larvae. We also show how this system can be employed as part of a new method for lineage tracing that would greatly facilitate the study of organogenesis and tissue regulation at any time in the life cycle. Conclusion This versatile and simple local heater has broad utility for the study of gene function and for lineage tracing. This system could be used to control hsp-driven gene expression in any organism simply by bringing the fiber optic tip in contact with the tissue of interest. Beyond these uses for the study of gene function, this device has wide-ranging utility in materials science and could easily be adapted for therapeutic purposes in humans.

  16. In vivo preclinical low field MRI monitoring of tumor growth following a suicide gene therapy in an ortho-topic mice model of human glioblastoma;Controle par IRM bas champ in vivo de l'efficacite d'une therapie genique par gene suicide dans un modele murin de glioblastome orthotopique

    Energy Technology Data Exchange (ETDEWEB)

    Breton, E.; Goetz, Ch.; Aubertin, G.; Constantinesco, A.; Choquet, Ph. [Service de biophysique et medecine nucleaire, hopital de Hautepierre, CHRU de Strasbourg, 67 - Strasbourg (France); Institut de mecanique des fluides et des solides, CNRS, universite de Strasbourg, 67 - Strasbourg (France); Kintz, J.; Accart, N.; Grellier, B.; Erbs, Ph.; Rooke, R. [Transgene SA, parc d' innovation, 67 - Illkirch Graffenstaden (France)

    2010-03-15

    Purpose The aim of this study was to monitor in vivo with low field MRI growth of a murine ortho-topic glioma model following a suicide gene therapy. Methods The gene therapy consisted in the stereotactic injection in the mice brain of a modified vaccinia virus Ankara (M.V.A.) vector encoding for a suicide gene (FCU1) that transforms a non toxic pro-drug 5-fluoro-cytosine (5-F.C.) to its highly cytotoxic derivatives 5-fluorouracil (5-F.U.) and 5-fluoro-uridine-5 monophosphate (5-F.U.M.P.). Using a warmed-up imaging cell, sequential 3D T1 and T2 0.1T MRI brain examinations were performed on 16 Swiss female nu/nu mice bearing ortho-topic human glioblastoma (U 87-MG cells). The 6-week in vivo MRI follow-up consisted in a weekly measurement of the intracerebral tumor volume leading to a total of 65 examinations. Mice were divided in four groups: sham group (n = 4), sham group treated with 5-F.C. only (n = 4), sham group with injection of M.V.A.-FCU1 vector only (n = 4), therapy group administered with M.V.A.-FCU1 vector and 5-F.C. (n = 4). Measurements of tumor volumes were obtained after manual segmentation of T1- and T2-weighted images. Results Intra-observer and inter-observer tumor volume measurements show no significant differences. No differences were found between T1 and T2 volume tumor doubling times between the three sham groups. A significant statistical difference (p < 0.05) in T1 and T2 volume tumor doubling times between the three sham groups and the animals treated with the intratumoral injection of M.V.A.-FCU1 vector in combination with 2 weeks per os 5-F.C. administration was demonstrated. Conclusion Preclinical low field MRI was able to monitor efficacy of suicide gene therapy in delaying the tumor growth in an in vivo mouse model of ortho-topic glioblastoma. (authors)

  17. Silencing of the pollen-specific gene NTP303 and its family members in tobacco affects in vivo pollen tube growth and results in male sterile plants.

    Science.gov (United States)

    de Groot, Peter; Weterings, Koen; de Been, Mark; Wittink, Floyd; Hulzink, Raymond; Custers, Jan; van Herpen, Marinus; Wullems, George

    2004-07-01

    In seed plants, successful fertilization requires correct regulation of pollen tube growth. At germination and during growth, the pollen tube interacts with tissues from the pistil while the pollen tube extends via tip growth. Despite the fact that much research has been devoted to the mechanisms regulating pollen tube growth, many aspects are currently unknown. Previously, we have isolated a pollen-specific gene from tobacco--NTP303--that probably functions during pollen tube growth. NTP303 is part of a family of five members. Its expression is regulated both at the transcriptional and at the translational level. While NTP303 transcripts accumulate to high levels between early bi-cellular and mature pollen stages, NTP303 protein is hardly detectable until germination and pollen tube growth. In order to elucidate the role and function of NTP303 in the pollen tube, we studied the effect of NTP303 gene silencing on pollen function. Therefore, we have transformed tobacco plants with NTP303 co-suppression and anti-sense gene constructs. In these plants, the kanamycin resistance trait--which was linked to the NTP303-silencing gene--was not transmitted through the male gametophyte. This indicated that lowering the transcript level of NTP303 and/or its family members interferes with pollen function. Because we could not find a readily distinguishable phenotype in pollen from the hemizygous anti-sense and co-suppression plants, we rescued the defective pollen to produce doubled haploid plants that were homozygous for the NTP303 anti-sense gene. We found that in pollen from these plants the transcript levels of all NTP303 family members were reduced. Although pollen and pollen tubes from these plants appeared completely normal in vitro, the pollen tubes showed slower growth rates in vivo and arrested in the style before they reached the ovary, so that fertilization failed. These data demonstrate that NTP303 and its family members are essential for normal pollen tube growth

  18. The gene expression of the neuronal protein, SLC38A9, changes in mouse brain after in vivo starvation and high-fat diet.

    Directory of Open Access Journals (Sweden)

    Sofie V Hellsten

    Full Text Available SLC38A9 is characterized as a lysosomal component of the amino acid sensing Ragulator-RAG GTPase complex, controlling the mechanistic target of rapamycin complex 1 (mTORC1. Here, immunohistochemistry was used to map SLC38A9 in mouse brain and staining was detected throughout the brain, in cortex, hypothalamus, thalamus, hippocampus, brainstem and cerebellum. More specifically, immunostaining was found in areas known to be involved in amino acid sensing and signaling pathways e.g. piriform cortex and hypothalamus. SLC38A9 immunoreactivity co-localized with both GABAergic and glutamatergic neurons, but not with astrocytes. SLC38A9 play a key role in the mTORC1 pathway, and therefore we performed in vivo starvation and high-fat diet studies, to measure gene expression alterations in specific brain tissues and in larger brain regions. Following starvation, Slc38a9 was upregulated in brainstem and cortex, and in anterior parts of the brain (Bregma 3.2 to -2.1mm. After high-fat diet, Slc38a9 was specifically upregulated in hypothalamus, while overall downregulation was noticed throughout the brain (Bregma 3.2 to -8.6mm.

  19. An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly.

    Science.gov (United States)

    Philippe, Lucas; Pandarakalam, George C; Fasimoye, Rotimi; Harrison, Neale; Connolly, Bernadette; Pettitt, Jonathan; Müller, Berndt

    2017-08-21

    Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Short hairpin RNA targeting 2B gene of coxsackievirus B3 exhibits potential antiviral effects both in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Yao Hailan

    2012-08-01

    Full Text Available Abstract Background Coxsackievirus B3 is an important infectious agent of viral myocarditis, pancreatitis and aseptic meningitis, but there are no specific antiviral therapeutic reagents in clinical use. RNA interference-based technology has been developed to prevent the viral infection. Methods To evaluate the impact of RNA interference on viral replication, cytopathogenicity and animal survival, short hairpin RNAs targeting the viral 2B region (shRNA-2B expressed by a recombinant vector (pGCL-2B or a recombinant lentivirus (Lenti-2B were tansfected in HeLa cells or transduced in mice infected with CVB3. Results ShRNA-2B exhibited a significant effect on inhibition of viral production in HeLa cells. Furthermore, shRNA-2B improved mouse survival rate, reduced the viral tissues titers and attenuated tissue damage compared with those of the shRNA-NC treated control group. Lenti-2B displayed more effective role in inhibition of viral replication than pGCL-2B in vivo. Conclusions Coxsackievirus B3 2B is an effective target of gene silencing against coxsackievirus B3 infection, suggesting that shRNA-2B is a potential agent for further development into a treatment for enterviral diseases.

  1. The Orf virus E3L homologue is able to complement deletion of the vaccinia virus E3L gene in vitro but not in vivo

    International Nuclear Information System (INIS)

    Vijaysri, Sangeetha; Talasela, Latha; Mercer, Andrew A.; Mcinnes, Colin J.; Jacobs, Bertram L.; Langland, Jeffrey O.

    2003-01-01

    Orf virus (OV), the prototypic parapoxvirus, is resistant to the effects of interferon (IFN) and this function of OV has been mapped to the OV20.0L gene. The protein product of this gene shares 31% amino acid identity to the E3L-encoded protein of vaccinia virus (VV) that is required for the broad host range and IFN-resistant phenotype of VV in cells in culture and for virulence of the virus in vivo. In this study we investigated whether the distantly related OV E3L homologue could complement the deletion of E3L in VV. The recombinant VV (VV/ORF-E3L) expressing the OV E3L homologue in place of VV E3L was indistinguishable from wt VV in its cell-culture phenotype. But VV/ORF-E3L was over a 1000-fold less pathogenic than wt VV (LD 50 > 5 x 10 6 PFU, compared to LD 50 of wtVV = 4 x 10 3 PFU) following intranasal infection of mice. While wt VV spread to the lungs and brain and replicated to high titers in the brain of infected mice, VV/ORF-E3L could not be detected in the lungs or brain following intranasal infection. VV/ORF-E3L was at least 100,000-fold less pathogenic than wt VV on intracranial injection. Domain swap experiments demonstrate that the difference in pathogenesis maps to the C-terminal domain of these proteins. This domain has been shown to be required for the dsRNA binding function of the VV E3L

  2. A monoallelic deletion of the TcCRT gene increases the attenuation of a cultured Trypanosoma cruzi strain, protecting against an in vivo virulent challenge.

    Directory of Open Access Journals (Sweden)

    Fernando J Sánchez-Valdéz

    2014-02-01

    Full Text Available Trypanosoma cruzi calreticulin (TcCRT is a virulence factor that binds complement C1, thus inhibiting the activation of the classical complement pathway and generating pro-phagocytic signals that increase parasite infectivity. In a previous work, we characterized a clonal cell line lacking one TcCRT allele (TcCRT+/- and another overexpressing it (TcCRT+, both derived from the attenuated TCC T. cruzi strain. The TcCRT+/- mutant was highly susceptible to killing by the complement machinery and presented a remarkable reduced propagation and differentiation rate both in vitro and in vivo. In this report, we have extended these studies to assess, in a mouse model of disease, the virulence, immunogenicity and safety of the mutant as an experimental vaccine. Balb/c mice were inoculated with TcCRT+/- parasites and followed-up during a 6-month period. Mutant parasites were not detected by sensitive techniques, even after mice immune suppression. Total anti-T. cruzi IgG levels were undetectable in TcCRT+/- inoculated mice and the genetic alteration was stable after long-term infection and it did not revert back to wild type form. Most importantly, immunization with TcCRT+/- parasites induces a highly protective response after challenge with a virulent T. cruzi strain, as evidenced by lower parasite density, mortality, spleen index and tissue inflammatory response. TcCRT+/- clones are restricted in two important properties conferred by TcCRT and indirectly by C1q: their ability to evade the host immune response and their virulence. Therefore, deletion of one copy of the TcCRT gene in the attenuated TCC strain generated a safe and irreversibly gene-deleted live attenuated parasite with high immunoprotective properties. Our results also contribute to endorse the important role of TcCRT as a T. cruzi virulence factor.

  3. Gene silencing of indoleamine 2,3-dioxygenase 2 in melanoma cells induces apoptosis through the suppression of NAD+ and inhibits in vivo tumor growth.

    Science.gov (United States)

    Liu, Yanling; Zhang, Yujuan; Zheng, Xiufen; Zhang, Xusheng; Wang, Hongmei; Li, Qin; Yuan, Keng; Zhou, Nanjing; Yu, Yanrong; Song, Na; Fu, Jiamin; Min, Weiping

    2016-05-31

    Indoleamine 2,3-dioxygenase 2 (IDO2) is a newly discovered enzyme that catalyzes the initial and rate-limiting step in the degradation of tryptophan. As a homologous protein of IDO1, IDO2 plays an inhibitory role in T cell proliferation, and it is essential for regulatory T cell (Treg) generation in healthy conditions. Little is known about the immune-independent functions of IDO2 relevant to its specific contributions to physiology and pathophysiology in cancer cells. The purpose of this study was to assess the impact of IDO2 gene silencing as a way to inhibit B16-BL6 cancer cells in a murine model. Here, for the first time, we show that knockdown of IDO2 using small interfering RNA (siRNA) inhibits cancer cell proliferation, arrests cell cycle in G1, induces greater cell apoptosis, and reduces cell migration in vitro. Knockdown of IDO2 decreased the generation of nicotinamide adenine dinucleotide (NAD+) while increasing the generation of reactive oxygen species (ROS). We further demonstrate that cell apoptosis, induced by IDO2 downregulation, can be weakened by addition of exogenous NAD+, suggesting a novel mechanism by which IDO2 promotes tumor growth through its metabolite product NAD+. In addition to in vitro findings, we also demonstrate that IDO2 silencing in tumor cells using short hairpin RNA (shRNA) delayed tumor formation and arrested tumor growth in vivo. In conclusion, this study demonstrates a new non-immune-associated mechanism of IDO2 in vitro and IDO2 expression in B16-BL6 cells contributes to cancer development and progression. Our research provides evidence of a novel target for gene silencing that has the potential to enhance cancer therapy.

  4. Tumorigenic potential of pituitary tumor transforming gene (PTTG in vivo investigated using a transgenic mouse model, and effects of cross breeding with p53 (+/− transgenic mice

    Directory of Open Access Journals (Sweden)

    Fong Miranda Y

    2012-11-01

    Full Text Available Abstract Background Pituitary tumor-transforming gene (PTTG is an oncogene that is overexpressed in variety of tumors and exhibits characteristics of a transforming gene. Previous transgenic mouse models to access the tumorigenic potential in the pituitary and ovary have resulted in dysplasia without formation of visible tumors, possibly due to the insufficient expression of PTTG. PTTG expression level is critical for ovarian tumorigenesis in a xenograft model. Therefore, the tumorigenic function of PTTG in vivo remains unclear. We generated a transgenic mouse that overexpresses PTTG driven by the CMV promoter to determine whether PTTG functions as a transforming oncogene that is capable of initiating tumorigenesis. Methods Transgenic animals were generated by microinjection of PTTG transgene into the male pronucleus of FVB 0.5 day old embryos. Expression levels of PTTG in tissues of transgenic animals were analyzed using an immunohistochemical analysis. H&E staining and immunohistostaining were performed to examine the type of tumor in transgenic and PTTG transgenic/p53+/- animals. Results PTTG transgenic offspring (TgPTTG were monitored for tumor development at various ages. H&E analysis was performed to identify the presence of cancer and hyperplastic conditions verified with the proliferation marker PCNA and the microvessel marker CD31. Immunohistochemistry was performed to determine transgene expression, revealing localization to the epithelium of the fallopian tube, with more generalized expression in the liver, lung, kidney, and spleen. At eight months of age, 2 out of 15 TgPTTG developed ovarian cancer, 2 out of 15 developed benign tumors, 2 out of 15 developed cervical dysplasia, and 3 out of 15 developed adenomyosis of the uterus. At ten months of age, 2 out of 10 TgPTTG developed adenocarcinoma of the ovary, 1 out of 10 developed a papillary serous adenocarcinoma, and 2 out of 10 presented with atypia of ovarian epithelial cells

  5. The tumor suppressors p33ING1 and p33ING2 interact with alien in vivo and enhance alien-mediated gene silencing.

    Science.gov (United States)

    Fegers, Inga; Kob, Robert; Eckey, Maren; Schmidt, Oliver; Goeman, Frauke; Papaioannou, Maria; Escher, Niko; von Eggeling, Ferdinand; Melle, Christian; Baniahmad, Aria

    2007-11-01

    The tumor suppressor p33ING1 is involved in DNA repair and cell cycle regulation. Furthermore, p33ING1 is a transcriptional silencer that recognizes the histone mark for trimethylated lysine 4 at histone H3. Interestingly, expression of p33ING1 and p33ING2 is able to induce premature senescence in primary human fibroblasts. The corepressor Alien is involved in gene silencing mediated by selected members of nuclear hormone receptors. In addition, Alien acts as a corepressor for E2F1, a member of the E2F cell cycle regulatory family. Furthermore, recent findings suggest that Alien is complexed with transcription factors participating in DNA repair and chromatin. Here, using a proteomic approach by surface-enhanced laser desorption ionization and mass spectrometry (SELDI-MS) combined with immunological techniques, we show that Alien interacts in vivo with the tumor suppressor p33ING1 as well as with the related tumor suppressor candidate p33ING2. The interaction of Alien with p33ING1 and p33ING2 was confirmed in vitro with GST-pull-down, suggesting a direct binding of Alien to these factors. The binding domain was mapped to a central region of Alien. Functionally, the expression of p33ING1 or p33ING2 enhances the Alien-mediated silencing, suggesting that the interaction plays a role in transcriptional regulation. Thus, the findings suggest that the identified interaction between Alien and the tumor suppressors p33ING1 and p33ING2 reveals a novel cellular protein network.

  6. Non-invasive in vivo imaging with radiolabelled FIAU for monitoring cancer gene therapy using herpes simplex virus type 1 thymidine kinase and ganciclovir

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Win-Ping; Lai, Wen-Fu [Graduate Institute of Biomedical Materials, Taipei Medical University, Taipei (Taiwan); Yang, Wen K.; Yang, Den-Mei [Institute of Biological Science, Academic Sinica, Taipei (Taiwan); Liu, Ren-Shyan [Department of Nuclear Medicine and National PET Cyclotron Center, Veterans General Hospital, Taipei (Taiwan); Hwang, Jeng-Jong; Wang, Hsin-Ell [Institute of Radiological Science, National Yang-Ming University, 155, Sec. 2, Lih-Nong Street, 112, Pei-tou, Taipei (Taiwan); Fu, Ying-Kai [Institute of Nuclear Energy, Atomic Energy Council, Taoyuan (Taiwan)

    2004-01-01

    in HSV1-tk(+) tumours (9.67%{+-}3.89%ID/g) than in HSV1-tk(-) tumours (0.48%{+-}0.19%ID/g). After seven consecutive daily treatments with the prodrug ganciclovir, planar gamma camera imaging showed HSV1-tk(+) tumour regression at day 4, and complete tumour regression at day 7. These results clearly demonstrate that the simplified n.c.a. synthesis process developed in this study is reliable and that the [{sup 131}I]FIAU product is useful for in vivo monitoring of HSV1-tk gene transfer, expression and gene therapy. (orig.)

  7. Influência de Lactobacillus rhamnosus na patogenicidade e na expressão de genes de virulência de Candida albicans: estudo in vitro e in vivo

    OpenAIRE

    Ribeiro, Felipe de Camargo [UNESP

    2015-01-01

    The high incidence of candidiasis caused by Candida albicans and the adaptability of this species, as well as resistance to antifungal drive the development of research on alternative therapies to control this infection. The aim of this study was to evaluate the influence of Lactobacillus rhamnosus and products of their metabolism against C. albicans, evaluating the pathogenicity and the expression of genes that regulate the formation of C. albicans biofilms in vitro and in vivo in invertebra...

  8. Gene transfer in rodents and primates as a new tool for modeling diseases in animals and assessing functions by in vivo imaging

    Energy Technology Data Exchange (ETDEWEB)

    Deglon, N. [Atomic Energy Commission (CEA), Dept. of Medical Research and MIRCen Program, 91 - Orsay (France)

    2006-07-01

    The identification of disease-causing genes in familial forms of neuro-degenerative disorders and the development of genetic models closely replicating human CNS pathologies have drastically changed our understanding of the molecular events leading to neuronal cell death. If these achievements open new opportunities of therapeutic interventions efficient delivery systems taking into account the specificity of the central nervous system are required to administer therapeutic candidates. In addition, there is a need to develop 1) genetic models in large animals that replicate late stages of the diseases and 2) imaging techniques suitable for longitudinal, quantitative and non-invasive evaluation of disease progression and the evaluation of new therapeutic strategies. Over the last few years, we have investigated the potential of lentiviral vectors as tool to model and treat CNS disorders. The use of lentiviral vectors to create animal model of these pathologies holds various advantages compared to classical transgenic approaches. Viral vectors are versatile, highly flexible tools to perform in vivo studies. Multiple genetic models can be created in a short period of time. High transduction efficiencies as well as robust and sustained trans-gene expression lead to the rapid appearance of functional and behavioral abnormalities and severe neuro-degeneration. Targeted injections in different brain areas can be used to investigate the regional specificity of the neuro-pathology and eliminate potential side effects associated with a widespread over-expression of the trans-gene. Finally, models can be established in different mammalian species including non-human primates, thereby providing an opportunity to assess complex behavioral changes and perform longitudinal follow-up of neuro-pathological alterations by imaging. We have demonstrated the proof of principle of this approach for Huntington's disease. We have shown that the intratriatal injection of lentiviral

  9. Gene transfer in rodents and primates as a new tool for modeling diseases in animals and assessing functions by in vivo imaging

    International Nuclear Information System (INIS)

    Deglon, N.

    2006-01-01

    The identification of disease-causing genes in familial forms of neuro-degenerative disorders and the development of genetic models closely replicating human CNS pathologies have drastically changed our understanding of the molecular events leading to neuronal cell death. If these achievements open new opportunities of therapeutic interventions efficient delivery systems taking into account the specificity of the central nervous system are required to administer therapeutic candidates. In addition, there is a need to develop 1) genetic models in large animals that replicate late stages of the diseases and 2) imaging techniques suitable for longitudinal, quantitative and non-invasive evaluation of disease progression and the evaluation of new therapeutic strategies. Over the last few years, we have investigated the potential of lentiviral vectors as tool to model and treat CNS disorders. The use of lentiviral vectors to create animal model of these pathologies holds various advantages compared to classical transgenic approaches. Viral vectors are versatile, highly flexible tools to perform in vivo studies. Multiple genetic models can be created in a short period of time. High transduction efficiencies as well as robust and sustained trans-gene expression lead to the rapid appearance of functional and behavioral abnormalities and severe neuro-degeneration. Targeted injections in different brain areas can be used to investigate the regional specificity of the neuro-pathology and eliminate potential side effects associated with a widespread over-expression of the trans-gene. Finally, models can be established in different mammalian species including non-human primates, thereby providing an opportunity to assess complex behavioral changes and perform longitudinal follow-up of neuro-pathological alterations by imaging. We have demonstrated the proof of principle of this approach for Huntington's disease. We have shown that the intratriatal injection of lentiviral vector

  10. Co-introduced functional CCR2 potentiates in vivo anti-lung cancer functionality mediated by T cells double gene-modified to express WT1-specific T-cell receptor.

    Directory of Open Access Journals (Sweden)

    Hiroaki Asai

    Full Text Available BACKGROUND AND PURPOSE: Although gene-modification of T cells to express tumor-related antigen-specific T-cell receptor (TCR or chimeric antigen receptor (CAR has clinically proved promise, there still remains room to improve the clinical efficacy of re-directed T-cell based antitumor adoptive therapy. In order to achieve more objective clinical responses using ex vivo-expanded tumor-responsive T cells, the infused T cells need to show adequate localized infiltration into the tumor. METHODOLOGY/PRINCIPAL FINDINGS: Human lung cancer cells variously express a tumor antigen, Wilms' Tumor gene product 1 (WT1, and an inflammatory chemokine, CCL2. However, CCR2, the relevant receptor for CCL2, is rarely expressed on activated T-lymphocytes. A HLA-A2402(+ human lung cancer cell line, LK79, which expresses high amounts of both CCL2 and WT1 mRNA, was employed as a target. Normal CD8(+ T cells were retrovirally gene-modified to express both CCR2 and HLA-A*2402-restricted and WT1(235-243 nonapeptide-specific TCR as an effector. Anti-tumor functionality mediated by these effector cells against LK79 cells was assessed both in vitro and in vivo. Finally the impact of CCL2 on WT1 epitope-responsive TCR signaling mediated by the effector cells was studied. Introduced CCR2 was functionally validated using gene-modified Jurkat cells and human CD3(+ T cells both in vitro and in vivo. Double gene-modified CD3(+ T cells successfully demonstrated both CCL2-tropic tumor trafficking and cytocidal reactivity against LK79 cells in vitro and in vivo. CCL2 augmented the WT1 epitope-responsive TCR signaling shown by relevant luciferase production in double gene-modified Jurkat/MA cells to express luciferase and WT1-specific TCR, and CCL2 also dose-dependently augmented WT1 epitope-responsive IFN-γ production and CD107a expression mediated by these double gene-modified CD3(+ T cells. CONCLUSION/SIGNIFICANCE: Introduction of the CCL2/CCR2 axis successfully potentiated in

  11. Co-Introduced Functional CCR2 Potentiates In Vivo Anti-Lung Cancer Functionality Mediated by T Cells Double Gene-Modified to Express WT1-Specific T-Cell Receptor

    Science.gov (United States)

    Asai, Hiroaki; Fujiwara, Hiroshi; An, Jun; Ochi, Toshiki; Miyazaki, Yukihiro; Nagai, Kozo; Okamoto, Sachiko; Mineno, Junichi; Kuzushima, Kiyotaka; Shiku, Hiroshi; Inoue, Hirofumi; Yasukawa, Masaki

    2013-01-01

    Background and Purpose Although gene-modification of T cells to express tumor-related antigen-specific T-cell receptor (TCR) or chimeric antigen receptor (CAR) has clinically proved promise, there still remains room to improve the clinical efficacy of re-directed T-cell based antitumor adoptive therapy. In order to achieve more objective clinical responses using ex vivo-expanded tumor-responsive T cells, the infused T cells need to show adequate localized infiltration into the tumor. Methodology/Principal Findings Human lung cancer cells variously express a tumor antigen, Wilms' Tumor gene product 1 (WT1), and an inflammatory chemokine, CCL2. However, CCR2, the relevant receptor for CCL2, is rarely expressed on activated T-lymphocytes. A HLA-A2402+ human lung cancer cell line, LK79, which expresses high amounts of both CCL2 and WT1 mRNA, was employed as a target. Normal CD8+ T cells were retrovirally gene-modified to express both CCR2 and HLA-A*2402-restricted and WT1235–243 nonapeptide-specific TCR as an effector. Anti-tumor functionality mediated by these effector cells against LK79 cells was assessed both in vitro and in vivo. Finally the impact of CCL2 on WT1 epitope-responsive TCR signaling mediated by the effector cells was studied. Introduced CCR2 was functionally validated using gene-modified Jurkat cells and human CD3+ T cells both in vitro and in vivo. Double gene-modified CD3+ T cells successfully demonstrated both CCL2-tropic tumor trafficking and cytocidal reactivity against LK79 cells in vitro and in vivo. CCL2 augmented the WT1 epitope-responsive TCR signaling shown by relevant luciferase production in double gene-modified Jurkat/MA cells to express luciferase and WT1-specific TCR, and CCL2 also dose-dependently augmented WT1 epitope-responsive IFN-γ production and CD107a expression mediated by these double gene-modifiedCD3+ T cells. Conclusion/Significance Introduction of the CCL2/CCR2 axis successfully potentiated in vivo anti-lung cancer

  12. Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    Directory of Open Access Journals (Sweden)

    Sha Ky

    2010-08-01

    Full Text Available Abstract Background Tissue differentiation is accompanied by genome-wide changes in the underlying chromatin structure and dynamics, or epigenome. By controlling when, where, and what regulatory factors have access to the underlying genomic DNA, the epigenome influences the cell's transcriptome and ultimately its function. Existing genomic methods for analyzing cell-type-specific changes in chromatin generally involve two elements: (i a source for purified cells (or nuclei of distinct types, and (ii a specific treatment that partitions or degrades chromatin by activity or structural features. For many cell types of great interest, such assays are limited by our inability to isolate the relevant cell populations in an organism or complex tissue containing an intertwined mixture of other cells. This limitation has confined available knowledge of chromatin dynamics to a narrow range of biological systems (cell types that can be sorted/separated/dissected in large numbers and tissue culture models or to amalgamations of diverse cell types (tissue chunks, whole organisms. Results Transgene-driven expression of DNA/chromatin modifying enzymes provides one opportunity to query chromatin structures in expression-defined cell subsets. In this work we combine in vivo expression of a bacterial DNA adenine methyltransferase (DAM with high throughput sequencing to sample tissue-specific chromatin accessibility on a genome-wide scale. We have applied the method (DALEC: Direct Asymmetric Ligation End Capture towards mapping a cell-type-specific view of genome accessibility as a function of differentiated state. Taking advantage of C. elegans strains expressing the DAM enzyme in diverse tissues (body wall muscle, gut, and hypodermis, our efforts yield a genome-wide dataset measuring chromatin accessibility at each of 538,000 DAM target sites in the C. elegans (diploid genome. Conclusions Validating the DALEC mapping results, we observe a strong association

  13. shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Qin Wang

    2014-01-01

    Full Text Available Colon cancer is one of the most common tumors of the digestive tract. Resistance to ionizing radiation (IR decreased therapeutic efficiency in these patients’ radiotherapy. XRCC2 is the key protein of DNA homologous recombination repair, and its high expression is associated with enhanced resistance to DNA damage induced by IR. Here, we investigated the effect of XRCC2 silencing on colon tumor cells’ growth and sensitivity to X-radiation in vitro and in vivo. Colon tumor cells (T84 cell line were cultivated in vitro and tumors originated from the cell line were propagated as xenografts in nude mice. The suppression of XRCC2 expression was achieved by using vector-based short hairpin RNA (shRNA in T84 cells. We found that the knockdown of XRCC2 expression effectively decreased T84 cellular proliferation and colony formation, and led to cell apoptosis and cell cycle arrested in G2/M phase induced by X-radiation in vitro. In addition, tumor xenograft studies suggested that XRCC2 silencing inhibited tumorigenicity after radiation treatment in vivo. Our data suggest that the suppression of XRCC2 expression rendered colon tumor cells more sensitive to radiation therapy in vitro and in vivo, implying XRCC2 as a promising therapeutic target for the treatment of radioresistant human colon cancer.

  14. Activator Gcn4 employs multiple segments of Med15/Gal11, including the KIX domain, to recruit mediator to target genes in vivo.

    Science.gov (United States)

    Jedidi, Iness; Zhang, Fan; Qiu, Hongfang; Stahl, Stephen J; Palmer, Ira; Kaufman, Joshua D; Nadaud, Philippe S; Mukherjee, Sujoy; Wingfield, Paul T; Jaroniec, Christopher P; Hinnebusch, Alan G

    2010-01-22

    Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4Delta cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.

  15. In Vitro and In Vivo Characterization of a Dual-Function Green Fluorescent Protein–HSV1-Thymidine Kinase Reporter Gene Driven by the Human Elongation Factor 1α Promoter

    Directory of Open Access Journals (Sweden)

    Gary D. Luker

    2002-04-01

    Full Text Available Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK as a reporter gene driven by the promoter for human elongation factor 1α (EF-1α-EGFP-TK. Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV. As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutylguanine ([18F]FHBG ≈ 8-[3H]penciclovir (8-[3H]PCV < 2′-fluoro-2′-deoxy-5-iodouracil-beta-d-arabinofuranoside (2-[14C]FIAU. Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.

  16. The in vivo biofilm

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Alhede, Maria; Alhede, Morten

    2013-01-01

    Bacteria can grow and proliferate either as single, independent cells or organized in aggregates commonly referred to as biofilms. When bacteria succeed in forming a biofilm within the human host, the infection often becomes very resistant to treatment and can develop into a chronic state. Biofilms...... have been studied for decades using various in vitro models, but it remains debatable whether such in vitro biofilms actually resemble in vivo biofilms in chronic infections. In vivo biofilms share several structural characteristics that differ from most in vitro biofilms. Additionally, the in vivo...... experimental time span and presence of host defenses differ from chronic infections and the chemical microenvironment of both in vivo and in vitro biofilms is seldom taken into account. In this review, we discuss why the current in vitro models of biofilms might be limited for describing infectious biofilms...

  17. Hyper-regulation of pyr-gene expression in Escherichia coli cells with slow ribosomes. Evidence for RNA polymerase pausing in vivo

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1988-01-01

    UTP-modulated attenuation of transcription is involved in regulating the synthesis of pyrimidine nucleotides in Escherichia coli. Thus, expression of two genes, pyrBI and pyrE, was shown to be under this type of control. The genes encode the two subunits of aspartate transcarbamylase and orotate...... transcription should terminate or continue into the structural genes. This paper described a study of pyrBI and pyrE gene regulation in cells where the ribosomes move slowly as a result of mutation in rpsL. It appears that expression of the two genes is hyper-regulated by the UTP pool in this type of cells...

  18. Activator Gcn4 Employs Multiple Segments of Med15/Gal11, Including the KIX Domain, to Recruit Mediator to Target Genes in Vivo*♦

    OpenAIRE

    Jedidi, Iness; Zhang, Fan; Qiu, Hongfang; Stahl, Stephen J.; Palmer, Ira; Kaufman, Joshua D.; Nadaud, Philippe S.; Mukherjee, Sujoy; Wingfield, Paul T.; Jaroniec, Christopher P.; Hinnebusch, Alan G.

    2009-01-01

    Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4Δ cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that i...

  19. Altered energy intake and the amplitude of the body temperature rhythm are associated with changes in phase, but not amplitude, of clock gene expression in the rat suprachiasmatic nucleus in vivo.

    Science.gov (United States)

    Goh, Grace H; Mark, Peter J; Maloney, Shane K

    2016-01-01

    Circadian rhythms in mammals are driven by a central clock in the suprachiasmatic nucleus (SCN). In vitro, temperature cycles within the physiological range can act as potent entraining cues for biological clocks. We altered the body temperature (Tc) rhythm in rats by manipulating energy intake (EI) to determine whether EI-induced changes in Tc oscillations are associated with changes in SCN clock gene rhythms in vivo. Male Wistar rats (n = 16 per diet) were maintained on either an ad libitum diet (CON), a high energy cafeteria diet (CAF), or a calorie restricted diet (CR), and Tc was recorded every 30 min for 6-7 weeks. SCN tissue was harvested from rats at zeitgeber time (ZT) 0, ZT6, ZT12, or ZT18. Expression of the clock genes Bmal1, Per2, Cry1, and Rev-erbα, the heat shock transcription factor Hsf1, and the heat shock protein Hsp90aa1, were determined using qPCR. The circadian profile of gene expression for each gene was characterized using cosinor analysis. Compared to the CON rats, the amplitude of Tc was decreased in CAF rats by 0.1 °C (p  0.25). Compared to CON, phase advances of the Tc, Bmal1, and Per2 rhythms were observed with CR feeding (p < 0.05), but CAF feeding elicited no significant changes in phase. The present results indicate that in vivo, the SCN is largely resistant to entrainment by EI-induced changes in the Tc rhythm, although some phase entrainment may occur.

  20. Desmosomes In Vivo

    Directory of Open Access Journals (Sweden)

    David Garrod

    2010-01-01

    Full Text Available The structure, function, and regulation of desmosomal adhesion in vivo are discussed. Most desmosomes in tissues exhibit calcium-independent adhesion, which is strongly adhesive or “hyperadhesive”. This is fundamental to tissue strength. Almost all studies in culture are done on weakly adhesive, calcium-dependent desmosomes, although hyperadhesion can be readily obtained in confluent cell culture. Calcium dependence is a default condition in vivo, found in wounds and embryonic development. Hyperadhesion appears to be associated with an ordered arrangement of the extracellular domains of the desmosomal cadherins, which gives rise to the intercellular midline identified in ultrastructural studies. This in turn probably depends on molecular order in the desmosomal plaque. Protein kinase C downregulates hyperadhesion and there is preliminary evidence that it may also be regulated by tyrosine kinases. Downregulation of desmosomes in vivo may occur by internalisation of whole desmosomes rather than disassembly. Hyperadhesion has implications for diseases such as pemphigus.

  1. Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo

    International Nuclear Information System (INIS)

    Rivera, Angel A.; Wang Minghui; Suzuki, Kaori; Uil, Taco G.; Krasnykh, Victor; Curiel, David T.; Nettelbeck, Dirk M.

    2004-01-01

    The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral oncolysis, to augment gene transfer compared with a nonreplicating adenoviral vector, or to combine virotherapy and gene therapy. Both the mode of transgene expression and the locale of transgene insertion into the virus genome critically determine the efficacy of this approach. We report here on the properties of oncolytic adenoviruses which contain the luciferase cDNA fused via an optimized internal ribosome entry site (IRES) to the immediate early adenoviral gene E1A (AdΔE1AIL), the early gene E2B (AdΔE2BIL), or the late fiber gene (AdΔfiberIL). These viruses showed distinct kinetics of transgene expression and luciferase activity. Early after infection, luciferase activities were lower for these viruses, especially for AdΔE2BIL, compared with nonreplicating AdTL, which contained the luciferase gene expressed from the strong CMV promoter. However, 6 days after infection, luciferase activities were approximately four (AdΔE1AIL) to six (AdΔfiberIL) orders of magnitude higher than for AdTL, reflecting virus replication and efficient transgene expression. Similar results were obtained in vivo after intratumoral injection of AdΔE2BIL, AdΔfiberIL, and AdTL. AdΔfiberIL and the parental virus, Ad5-Δ24, resulted in similar cytotoxicity, but AdΔE2BIL and AdΔE1AIL were slightly attenuated. Disruption of the expression of neighboring viral genes by insertion of the transgene was minimal for AdΔE2BIL and AdΔfiberIL, but substantial for AdΔE1AIL. Our observations suggest that insertion of IRES-transgene cassettes into viral transcription units is an attractive strategy for the development of armed oncolytic adenoviruses with defined kinetics and strength of transgene expression

  2. Vitamin D metabolism and effects on pluripotency genes and cell differentiation in testicular germ cell tumors in vitro and in vivo

    DEFF Research Database (Denmark)

    Blomberg Jensen, Martin; Jørgensen, Anne; Nielsen, John Erik

    2012-01-01

    and express pluripotency factors (NANOG/OCT4). Vitamin D (VD) is metabolized in the testes, and here, we examined VD metabolism in TGCT differentiation and pluripotency regulation. We established that the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human fetal germ cells, CIS, and invasive......) treatment in vivo. These novel findings show that VD metabolism is involved in the mesodermal transition during differentiation of cancer cells with embryonic stem cell characteristics, which points to a function for VD during early embryonic development and possibly in the pathogenesis of TGCTs.......Testicular germ cell tumors (TGCTs) are classified as either seminomas or nonseminomas. Both tumors originate from carcinoma in situ (CIS) cells, which are derived from transformed fetal gonocytes. CIS, seminoma, and the undifferentiated embryonal carcinoma (EC) retain an embryonic phenotype...

  3. Differential binding of Lef1 and Msx1/2 transcription factors to Dkk1 CNEs correlates with reporter gene expression in vivo.

    Directory of Open Access Journals (Sweden)

    Oliver Lieven

    Full Text Available Besides the active Wnt signalling itself, the extracellular inhibition by Dkk1 is important for various embryonic developmental processes, such as optic vesicle differentiation and facial outgrowth. Although a feedback crosstalk of the active Wnt/β-catenin signaling and Dkk1 regulation has been suggested, the control of Dkk1 transcription by the Tcf/Lef1 mediated Wnt signalling and its connection to additional signalling factors has not been elucidated in vivo. Here, we used a combination of transgenic mouse approaches and biochemical analyses to unravel the direct Dkk1 transcriptional regulation via Tcf/Lefs. By using site directed mutagenesis, we tested several conserved Tcf/Lef1 binding sites within Dkk1 conserved non-coding elements (CNEs and found that these are required for tissue specific reporter expression. In addition a conserved Msx1/2 binding site is required for retinal reporter expression and Msx2 but not Msx1 binds its conserved binding site within CNE195 in the optic cups. Within craniofacial expression domains, Lef1 interferes with Dkk1 directly via two conserved Tcf/Lef1 binding sites in the craniofacial enhancer CNE114, both of which are required for the general craniofacial Dkk1 reporter activation. Furthermore, these Tcf/Lef1 sites are commonly bound in the whisker hair bud mesenchyme but specifically Tcf/Lef1 (no. 2 is required for mandibular activation and repression of maxillar Dkk1 activation. Lastly, we tested the Tcf/Lef1 binding capacities of the Dkk1 promoter and found that although Lef1 binds the Dkk1 promoter, these sites are not sufficient for tissue specific Dkk1 activation. Together, we here present the importance of conserved Tcf/Lef1 and Msx1/2 sites that are required for differential Dkk1 transcriptional reporter activation in vivo. This requirement directly correlates with Lef1 and Msx1/2 interaction with these genomic loci.

  4. In vitro and in vivo gene therapy with CMV vector-mediated presumed dog beta-nerve growth factor in pyridoxine-induced neuropathy dogs.

    Science.gov (United States)

    Chung, Jin Young; Choi, Jung Hoon; Shin, Il Seob; Choi, Eun Wha; Hwang, Cheol Yong; Lee, Sang Koo; Youn, Hwa Young

    2008-12-01

    Due to the therapeutic potential of gene therapy for neuronal injury, many studies of neurotrophic factors, vectors, and animal models have been performed. The presumed dog beta-nerve growth factor (pdbeta-NGF) was generated and cloned and its expression was confirmed in CHO cells. The recombinant pdbeta-NGF protein reacted with a human beta-NGF antibody and showed bioactivity in PC12 cells. The pdbeta-NGF was shown to have similar bioactivity to the dog beta-NGF. The recombinant pdbeta-NGF plasmid was administrated into the intrathecal space in the gene therapy group. Twenty-four hours after the vector inoculation, the gene therapy group and the positive control group were intoxicated with excess pyridoxine for seven days. Each morning throughout the test period, the dogs' body weight was taken and postural reaction assessments were made. Electrophysiological recordings were performed twice, once before the experiment and once after the test period. After the experimental period, histological analysis was performed. Dogs in the gene therapy group had no weight change and were normal in postural reaction assessments. Electrophysiological recordings were also normal for the gene therapy group. Histological analysis showed that neither the axons nor the myelin of the dorsal funiculus of L4 were severely damaged in the gene therapy group. In addition, the dorsal root ganglia of L4 and the peripheral nerves (sciatic nerve) did not experience severe degenerative changes in the gene therapy group. This study is the first to show the protective effect of NGF gene therapy in a dog model.

  5. In vivo release of calcitonin gene-related peptide-like material from the cervicotrigeminal area in the rat. Effects of electrical and noxious stimulations of the muzzle.

    Science.gov (United States)

    Pohl, M; Collin, E; Bourgoin, S; Clot, A M; Hamon, M; Cesselin, F; Le Bars, D

    1992-10-01

    The continuous perfusion with an artificial cerebrospinal fluid of the cervicotrigeminal area of the spinal cord in halothane-anaesthetized rats allowed the collection of calcitonin gene-related peptide-like material with the same immunological and chromatographic characteristics as authentic rat alpha-calcitonin gene-related peptide. The spinal release of calcitonin gene-related peptide-like material could be significantly increased by the local application of 60 mM K+ (approximately +100%), high-intensity percutaneous electrical stimulation (approximately +200%) and noxious heat (by immersion in water at 52 degrees C; approximately +150%) applied to the muzzle. By contrast, noxious mechanical (pinches) and chemical (subcutaneous formalin injection) stimulations and deep cooling (by immersion in water at 0 degrees C) of the muzzle did not alter the spinal release of calcitonin gene-related peptide-like material. In addition, low-intensity electrical stimulation, recruiting only the A alpha/beta primary afferent fibres, significantly reduced (approximately -30%) the release of calcitonin gene-related peptide-like material from the cervicotrigeminal area. These data suggest that among the various types of natural noxious stimuli, noxious heat may selectively excite calcitonin gene-related peptide-containing A delta and C primary afferent fibres projecting within the dorsal horn of the spinal cord, and that activation of A alpha/beta fibres reduces spontaneous calcitonin gene-related peptide-like material release possibly through an inhibitory presynaptic control of calcitonin gene-related peptide-containing A delta/C fibres.

  6. Direct visualization of electroporation-assisted in vivo gene delivery to tumors using intravital microscopy – spatial and time dependent distribution

    Directory of Open Access Journals (Sweden)

    Dachs Gabi U

    2004-11-01

    Full Text Available Abstract Background Electroporation is currently receiving much attention as a way to increase drug and DNA delivery. Recent studies demonstrated the feasibility of electrogene therapy using a range of therapeutic genes for the treatment of experimental tumors. However, the transfection efficiency of electroporation-assisted DNA delivery is still low compared to viral methods and there is a clear need to optimize this approach. In order to optimize treatment, knowledge about spatial and time dependency of gene expression following delivery is of utmost importance in order to improve gene delivery. Intravital microscopy of tumors growing in dorsal skin fold window chambers is a useful method for monitoring gene transfection, since it allows non-invasive dynamic monitoring of gene expression in tumors in a live animal. Methods Intravital microscopy was used to monitor real time spatial distribution of the green fluorescent protein (GFP and time dependence of transfection efficiency in syngeneic P22 rat tumor model. DNA alone, liposome-DNA complexes and electroporation-assisted DNA delivery using two different sets of electric pulse parameters were compared. Results Electroporation-assisted DNA delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz was superior to other methods and resulted in 22% increase in fluorescence intensity in the tumors up to 6 days post-transfection, compared to the non-transfected area in granulation tissue. Functional GFP was detected within 5 h after transfection. Cells expressing GFP were detected throughout the tumor, but not in the surrounding tissue that was not exposed to electric pulses. Conclusions Intravital microscopy was demonstrated to be a suitable method for monitoring time and spatial distribution of gene expression in experimental tumors and provided evidence that electroporation-assisted gene delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz is an effective method, resulting in early onset and homogenous

  7. Direct visualization of electroporation-assisted in vivo gene delivery to tumors using intravital microscopy – spatial and time dependent distribution

    International Nuclear Information System (INIS)

    Cemazar, Maja; Wilson, Ian; Dachs, Gabi U; Tozer, Gillian M; Sersa, Gregor

    2004-01-01

    Electroporation is currently receiving much attention as a way to increase drug and DNA delivery. Recent studies demonstrated the feasibility of electrogene therapy using a range of therapeutic genes for the treatment of experimental tumors. However, the transfection efficiency of electroporation-assisted DNA delivery is still low compared to viral methods and there is a clear need to optimize this approach. In order to optimize treatment, knowledge about spatial and time dependency of gene expression following delivery is of utmost importance in order to improve gene delivery. Intravital microscopy of tumors growing in dorsal skin fold window chambers is a useful method for monitoring gene transfection, since it allows non-invasive dynamic monitoring of gene expression in tumors in a live animal. Intravital microscopy was used to monitor real time spatial distribution of the green fluorescent protein (GFP) and time dependence of transfection efficiency in syngeneic P22 rat tumor model. DNA alone, liposome-DNA complexes and electroporation-assisted DNA delivery using two different sets of electric pulse parameters were compared. Electroporation-assisted DNA delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz was superior to other methods and resulted in 22% increase in fluorescence intensity in the tumors up to 6 days post-transfection, compared to the non-transfected area in granulation tissue. Functional GFP was detected within 5 h after transfection. Cells expressing GFP were detected throughout the tumor, but not in the surrounding tissue that was not exposed to electric pulses. Intravital microscopy was demonstrated to be a suitable method for monitoring time and spatial distribution of gene expression in experimental tumors and provided evidence that electroporation-assisted gene delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz is an effective method, resulting in early onset and homogenous distribution of gene expression in the syngeneic P22 rat tumor model

  8. Preparation of a Nanoscaled Poly(vinyl alcohol/Hydroxyapatite/DNA Complex Using High Hydrostatic Pressure Technology for In Vitro and In Vivo Gene Delivery

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Kimura

    2011-01-01

    Full Text Available Our previous research showed that poly(vinyl alcohol (PVA nanoparticles incorporating DNA with hydrogen bonds obtained by high hydrostatic pressurization are able to deliver DNA without any significant cytotoxicity. To enhance transfection efficiency of PVA/DNA nanoparticles, we describe a novel method to prepare PVA/DNA nanoparticles encapsulating nanoscaled hydroxyapatites (HAps prepared by high hydrostatic pressurization (980 MPa, which is designed to facilitate endosomal escape induced by dissolving HAps in an endosome. Scanning electron microscopic observation and dynamic light scattering measurement revealed that HAps were significantly encapsulated in PVA/HAp/DNA nanoparticles. The cytotoxicity, cellular uptake, and transgene expression of PVA/HAp/DNA nanoparticles were investigated using COS-7 cells. It was found that, in contrast to PVA/DNA nanoparticles, their internalization and transgene expression increased without cytotoxicity occurring. Furthermore, a similar level of transgene expression between plasmid DNA and PVA/HAp/DNA nanoparticles was achieved using in vivo hydrodynamic injection. Our results show a novel method of preparing PVA/DNA nanoparticles encapsulating HAp nano-crystals by using high hydrostatic pressure technology and the potential use of HAps as an enhancer of the transfection efficiency of PVA/DNA nanoparticles without significant cytotoxicity.

  9. 8-methoxypsoralen and ultraviolet A radiation activate the human elastin promoter in transgenic mice: in vivo and in vitro evidence for gene induction

    International Nuclear Information System (INIS)

    Bernstein, E.F.; Brown, D.B.; Takeuchi, Tsunemichi; Kong, S.K.; Uitto, Jouni; Gasparro, F.P.

    1996-01-01

    Treatment of skin diseases with the combination of 8-methoxypsoralen and ultraviolet A radiation (PUVA) results in clinical alterations in treated skin that resemble those observed in chronically photodamaged skin. The PUVA-treated patients develop nonmelanoma skin cancers, pigmentary alterations and wrinkling characteristic of sun-induced changes. The major alteration in the dermis of sun-damaged skin is the deposition of abnormal elastic fibers, termed solar elastosis. Up-regulation of elastin promoter activity in dermal fibroblasts explains the excess elastic tissue but not the reason for the aberrant morphology of the elastotic material. In order to study photoaging in an experimental system we utilized a transgenic mouse line that expresses the human elastin promoter/chloramphenicol acetyltransferase construct in a tissue-specific and developmentally regulated manner. Although UVB radiation has been demonstrated to increase promoter activity in vitro, UVA fails to demonstrate a similar effect at the doses utilized. In this study, we demonstrate the ability of PUVA treatment to up regulate elastin promoter activity both in vitro and in vivo. These data help to explain the development of photoaging in sun-protected PUVA-treated skin. We attribute the up-regulation of elastin promoter activity in response to PUVA to the formation of DNA photoadducts, which do not occur in response to UVA radiation alone. (UK)

  10. In vivo changes of hemopoietic progenitors and the expression of the interleukin 5 gene in eosinophilic mice infected with Toxocara canis.

    Science.gov (United States)

    Yamaguchi, Y; Matsui, T; Kasahara, T; Etoh, S; Tominaga, A; Takatsu, K; Miura, Y; Suda, T

    1990-12-01

    It has been demonstrated that purified recombinant interleukin 5 (rIL-5) supports the terminal differentiation and proliferation of eosinophilic precursors in vitro and plays an important role in increasing the functional activities of eosinophils. In this study, we examined the hemopoietic changes and analyzed murine (m) IL-5 mRNA expression in eosinophilic mice infected with the helminth Toxocara canis. In eosinophilic mice, eosinophils increased in number in both bone marrow and spleen. However, the number of eosinophilic precursors increased markedly in spleen cells of eosinophilic mice but remained relatively constant in the bone marrow. In the presence of granulocyte colony-stimulating factor (G-CSF), the number of granulocytic precursors increased in the spleen cells of eosinophilic mice. From these findings, the condition of eosinophilopoiesis in eosinophilic mice is accompanied by an increase in granulocyte-macrophage progenitors as well as eosinophil progenitors. Using Northern blot analysis, a weak but definite band corresponding to mIL-5 mRNA was detected in spleen cells of mice 4 and 5 days after helminthic infection. In addition, these data were confirmed by in vitro polymerase chain reaction (PCR) amplification of mRNA obtained from these spleen cells. Finally, injections of a monoclonal antibody against mIL-5 completely suppressed the blood eosinophilia in mice infected with T. canis. In conclusion, IL-5 is suggested to play a major role in eosinophilopoiesis in vivo.

  11. Gene expression profile of the cartilage tissue spontaneously regenerated in vivo by using a novel double-network gel: Comparisons with the normal articular cartilage

    Directory of Open Access Journals (Sweden)

    Kurokawa Takayuki

    2011-09-01

    Full Text Available Abstract Background We have recently found a phenomenon that spontaneous regeneration of a hyaline cartilage-like tissue can be induced in a large osteochondral defect by implanting a double-network (DN hydrogel plug, which was composed of poly-(2-Acrylamido-2-methylpropanesulfonic acid and poly-(N, N'-Dimetyl acrylamide, at the bottom of the defect. The purpose of this study was to clarify gene expression profile of the regenerated tissue in comparison with that of the normal articular cartilage. Methods We created a cylindrical osteochondral defect in the rabbit femoral grooves. Then, we implanted the DN gel plug at the bottom of the defect. At 2 and 4 weeks after surgery, the regenerated tissue was analyzed using DNA microarray and immunohistochemical examinations. Results The gene expression profiles of the regenerated tissues were macroscopically similar to the normal cartilage, but showed some minor differences. The expression degree of COL2A1, COL1A2, COL10A1, DCN, FMOD, SPARC, FLOD2, CHAD, CTGF, and COMP genes was greater in the regenerated tissue than in the normal cartilage. The top 30 genes that expressed 5 times or more in the regenerated tissue as compared with the normal cartilage included type-2 collagen, type-10 collagen, FN, vimentin, COMP, EF1alpha, TFCP2, and GAPDH genes. Conclusions The tissue regenerated by using the DN gel was genetically similar but not completely identical to articular cartilage. The genetic data shown in this study are useful for future studies to identify specific genes involved in spontaneous cartilage regeneration.

  12. Senescent vs. non-senescent cells in the human annulus in vivo: Cell harvest with laser capture microdissection and gene expression studies with microarray analysis

    Directory of Open Access Journals (Sweden)

    Ingram Jane A

    2010-01-01

    Full Text Available Abstract Background Senescent cells are well-recognized in the aging/degenerating human disc. Senescent cells are viable, cannot divide, remain metabolically active and accumulate within the disc over time. Molecular analysis of senescent cells in tissue offers a special challenge since there are no cell surface markers for senescence which would let one use fluorescence-activated cell sorting as a method for separating out senescent cells. Methods We employed a novel laser capture microdissection (LCM design to selectively harvest senescent and non-senescent annulus cells in paraffin-embedded tissue, and compared their gene expression with microarray analysis. LCM was used to separately harvest senescent and non-senescent cells from 11 human annulus specimens. Results Microarray analysis revealed significant differences in expression levels in senescent cells vs non-senescent cells: 292 genes were upregulated, and 321 downregulated. Genes with established relationships to senescence were found to be significantly upregulated in senescent cells vs. non-senescent cells: p38 (MPAK14, RB-Associated KRAB zinc finger, Discoidin, CUB and LCCL domain, growth arrest and DNA-damage inducible beta, p28ING5, sphingosine-1-phosphate receptor 2 and somatostatin receptor 3; cyclin-dependent kinase 8 showed significant downregulation in senescent cells. Nitric oxidase synthase 1, and heat shock 70 kDa protein 6, both of which were significantly down-regulated in senescent cells, also showed significant changes. Additional genes related to cytokines, cell proliferation, and other processes were also identified. Conclusions Our LCM-microarray analyses identified a set of genes associated with senescence which were significantly upregulated in senescent vs non-senescent cells in the human annulus. These genes include p38 MAP kinase, discoidin, inhibitor of growth family member 5, and growth arrest and DNA-damage-inducible beta. Other genes, including genes

  13. Regional brain metabolite abnormalities in inherited prion disease and asymptomatic gene carriers demonstrated in vivo by quantitative proton magnetic resonance spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Waldman, A.D.; Cordery, R.J.; Godbolt, A.; Rossor, M.N. [University College London, Dementia Research Group, Department of Neurodegenerative Disease, Institute of Neurology, London (United Kingdom); Imperial College of Science, Technology and Medicine, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, London (United Kingdom); MacManus, D.G. [University College London, NMR Research Unit, Department of Clinical Neurology, Institute of Neurology, London (United Kingdom); Collinge, J. [University College London, MRC Prion Unit, Department of Neurodegenerative Disease, Institute of Neurology, London (United Kingdom)

    2006-06-15

    Inherited prion diseases are caused by mutations in the gene which codes for prion protein (PrP), leading to proliferation of abnormal PrP isomers in the brain and neurodegeneration; they include Gerstmann-Straeussler-Scheinker disease (GSS), fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). We studied two patients with symptomatic inherited prion disease (P102L) and two pre-symptomatic P102L gene carriers using quantitative magnetic resonance spectroscopy (MRS). Short echo time spectra were acquired from the thalamus, caudate region and frontal white matter, metabolite levels and ratios were measured and z-scores calculated for individual patients relative to age-matched normal controls. MRS data were compared with structural magnetic resonance imaging. One fCJD case had generalised atrophy and showed increased levels of myo-inositol (MI) in the thalamus (z=3.7). The other had decreased levels of N-acetylaspartate (z=4) and diffuse signal abnormality in the frontal white matter. Both asymptomatic gene carriers had normal imaging, but increased frontal white matter MI (z=4.3, 4.1), and one also had increased MI in the caudate (z=5.3). Isolated MI abnormalities in asymptomatic gene carriers are a novel finding and may reflect early glial proliferation, prior to significant neuronal damage. MRS provides potential non-invasive surrogate markers of early disease and progression in inherited prion disease. (orig.)

  14. Regional brain metabolite abnormalities in inherited prion disease and asymptomatic gene carriers demonstrated in vivo by quantitative proton magnetic resonance spectroscopy

    International Nuclear Information System (INIS)

    Waldman, A.D.; Cordery, R.J.; Godbolt, A.; Rossor, M.N.; MacManus, D.G.; Collinge, J.

    2006-01-01

    Inherited prion diseases are caused by mutations in the gene which codes for prion protein (PrP), leading to proliferation of abnormal PrP isomers in the brain and neurodegeneration; they include Gerstmann-Straeussler-Scheinker disease (GSS), fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). We studied two patients with symptomatic inherited prion disease (P102L) and two pre-symptomatic P102L gene carriers using quantitative magnetic resonance spectroscopy (MRS). Short echo time spectra were acquired from the thalamus, caudate region and frontal white matter, metabolite levels and ratios were measured and z-scores calculated for individual patients relative to age-matched normal controls. MRS data were compared with structural magnetic resonance imaging. One fCJD case had generalised atrophy and showed increased levels of myo-inositol (MI) in the thalamus (z=3.7). The other had decreased levels of N-acetylaspartate (z=4) and diffuse signal abnormality in the frontal white matter. Both asymptomatic gene carriers had normal imaging, but increased frontal white matter MI (z=4.3, 4.1), and one also had increased MI in the caudate (z=5.3). Isolated MI abnormalities in asymptomatic gene carriers are a novel finding and may reflect early glial proliferation, prior to significant neuronal damage. MRS provides potential non-invasive surrogate markers of early disease and progression in inherited prion disease. (orig.)

  15. In vivo effect of an antilipolytic drug (3,5'-dimethylpyrazole) on autophagic proteolysis and autophagy-related gene expression in rat liver

    International Nuclear Information System (INIS)

    Donati, Alessio; Ventruti, Annamaria; Cavallini, Gabriella; Masini, Matilde; Vittorini, Simona; Chantret, Isabelle; Codogno, Patrice; Bergamini, Ettore

    2008-01-01

    Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455-470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5'-dimethylpyrazole, DMP, 12 mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague-Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autophagy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis

  16. From In Vivo to In Vitro: Dynamic Analysis of Plasmodium falciparum var Gene Expression Patterns of Patient Isolates during Adaptation to Culture

    Science.gov (United States)

    Huang, Yufu; Xue, Xiangyang; Yan, He; Sun, Xiaodong; Wang, Jian; McCutchan, Thomas F.; Pan, Weiqing

    2011-01-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, plays a crucial role in disease virulence through its involvement in binding to various host cellular receptors during infection. Growing evidence suggests that differential expression of the various var subgroups may be involved in parasite virulence. To further explore this issue, we have collected isolates from symptomatic patients in south China-Myanmar border, and characterized their sequence diversity and transcription profiles over time of var gene family, and cytoadherence properties from the time of their initial collection and extending through a two month period of adaptation to culture. Initially, we established a highly diverse, DBLα (4 cysteines) subtype-enriched, but unique local repertoire of var-DBL1α sequences by cDNA cloning and sequencing. Next we observed a rapid transcriptional decline of upsA- and upsB-subtype var genes at ring stage through qRT-PCR assays, and a switching event from initial ICAM-I binding to the CD36-binding activity during the first week of adaptive cultivation in vitro. Moreover, predominant transcription of upsA var genes was observed to be correlated with those isolates that showed a higher parasitemia at the time of collection and the ICAM-1-binding phenotype in culture. Taken together, these data indicate that the initial stage of adaptive process in vitro significantly influences the transcription of virulence-related var subtypes and expression of PfEMP1 variants. Further, the specific upregulation of the upsA var genes is likely linked to the rapid propagation of the parasite during natural infection due to the A-type PfEMP1 variant-mediated growth advantages. PMID:21674009

  17. In vivo Exposure Effects of 99mTc-methoxyisobutylisonitrile on the FDXR and XPA Genes Expression in Human Peripheral Blood Lymphocytes

    Directory of Open Access Journals (Sweden)

    Mohammad Taghi Bahreyni-Toossi

    2018-01-01

    Full Text Available Objective(s: In recent years, the application of radiopharmaceuticals in nuclear medicine has increased substantially. Following the diagnostic procedures performed in nuclear medicine departments, such as myocardial perfusion imaging, patients generally receive considerable doses of radiation. Normally, radiation-induced DNA damages are expected following exposure to a low-dose ionizing radiation. In order to detect molecular changes, high-sensitivity techniques must be utilized. The aim of this study was to assess the effect of a low-dose (below 10 mSv gamma ray on gene expression using quantitative real-time polymerase chain reaction (qRT-PCR. Methods: Blood samples were obtained from 20 volunteer patients who underwent myocardial perfusion imaging. They were given various doses of Technetium99-m methoxyisobutylisonitrile (99mTc-MIBI. After that, peripheral blood mononuclear cells (PBMNs were derived, and then total RNA was extracted and reverse-transcribed to cDNA. Finally, the expression levels of xeroderma pigmentosum complementation group-A (XPA and ferredoxin reductase (FDXR genes were determinded through qRT-PCR technique using SYBR Green. Results: XPA and FDXR expression levels were obtained following a very low-dose ionizing radiation. A significant up-regulation of both genes was observed, and the gene expression level of each individual patient was different. If differences in the administered activity and radiosensitivity are taken into account, the observed differences could be justified. Furthermore, gender and age did not play a significant role in the expression levels of the genes under study. Conclusion: The up-regulation of FDXR after irradiation revealed the high-sensitivity level of this gene; therefore, it could be used as an appropriate biomarker for biological dosimetry. On the other hand, the up-regulation of XPA is an indication of DNA repair following radiation exposure. According to linear no-threshold model (LNT

  18. Development of a gene therapy strategy to target hepatocellular carcinoma based inhibition of protein phosphatase 2A using the α-fetoprotein promoter enhancer and pgk promoter: an in vitro and in vivo study

    International Nuclear Information System (INIS)

    Li, Wei; Tao, Min; Li, Dao-Ming; Chen, Kai; Chen, Zheng; Zong, Yang; Yin, Hong; Xu, Ze-Kuan; Zhu, Yi; Gong, Fei-Ran

    2012-01-01

    Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Current therapies are insufficient, making HCC an intractable disease. Our previous studies confirmed that inhibition of protein phosphatase 2A (PP2A) may provide a promising therapeutic strategy for cancer. Unfortunately, constitutive expression of PP2A in normal tissues limits the application of PP2A inhibition. Thus, a HCC-specific gene delivery system should be developed. The α-fetoprotein (AFP) promoter is commonly used in HCC-specific gene therapy strategies; however, the utility of this approach is limited due to the weak activity of the AFP promoter. It has been shown that linking the AFP enhancer with the promoter of the non-tissue-specific, human housekeeping phosphoglycerate kinase (pgk) gene can generate a strong and HCC-selective promoter. We constructed a HCC-specific gene therapy system to target PP2A using the AFP enhancer/pgk promoter, and evaluated the efficiency and specificity of this system both in vitro and in vivo. AFP enhancer/pgk promoter-driven expression of the dominant negative form of the PP2A catalytic subunit α (DN-PP2Acα) exerted cytotoxic effects against an AFP-positive human hepatoma cell lines (HepG2 and Hep3B), but did not affect AFP-negative human hepatoma cells (SK-HEP-1) or normal human liver cells (L-02). Moreover, AFP enhancer/pgk promoter driven expression of DN-PP2Acα inhibited the growth of AFP-positive HepG2 tumors in nude mice bearing solid tumor xenografts, but did not affect AFP-negative SK-HEP-1 tumors. The novel approach of AFP enhancer/pgk promoter-driven expression of DN-PP2Acα may provide a useful cancer gene therapy strategy to selectively target HCC

  19. The Cyclic Di-GMP Phosphodiesterase Gene Rv1357c/BCG1419c Affects BCG Pellicle Production and In Vivo Maintenance.

    Science.gov (United States)

    Flores-Valdez, Mario Alberto; Aceves-Sánchez, Michel de Jesús; Pedroza-Roldán, César; Vega-Domínguez, Perla Jazmín; Prado-Montes de Oca, Ernesto; Bravo-Madrigal, Jorge; Laval, Françoise; Daffé, Mamadou; Koestler, Ben; Waters, Christopher M

    2015-02-01

    Bacteria living in a surface-attached community that contains a heterogeneous population, coated with an extracellular matrix, and showing drug tolerance (biofilms) are often linked to chronic infections. In mycobacteria, the pellicle mode of growth has been equated to an in vitro biofilm and meets several of the criteria mentioned above, while tuberculosis infection presents a chronic (latent) phase of infection. As mycobacteria lack most genes required to control biofilm production by other microorganisms, we deleted or expressed from the hsp60 strong promoter the only known c-di-GMP phosphodiesterase (PDE) gene in Mycobacterium bovis BCG. We found changes in pellicle production, cellular protein profiles, lipid production, resistance to nitrosative stress and maintenance in lungs and spleens of immunocompetent BALB/mice. Our results show that pellicle production and capacity to remain within the host are linked in BCG. © 2015 International Union of Biochemistry and Molecular Biology.

  20. In vivo effects of UV radiation on multiple endpoints and expression profiles of DNA repair and heat shock protein (Hsp) genes in the cycloid copepod Paracyclopina nana

    Energy Technology Data Exchange (ETDEWEB)

    Won, Eun-Ji; Han, Jeonghoon [Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Lee, Yeonjung; Kumar, K. Suresh; Shin, Kyung-Hoon [Department of Marine Sciences and Convergent Technology, College of Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Lee, Su-Jae [Department of Life Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Park, Heum Gi, E-mail: hgpark@gwnu.ac.kr [Department of Marine Resource Development, College of Life Sciences, Gangneung-Wonju National University, Gangneung 210-702 (Korea, Republic of); Lee, Jae-Seong, E-mail: jslee2@skku.edu [Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2015-08-15

    Highlights: • UV-B radiation induced a significant reduction of the re-brooding rate of ovigerous females. • A dose-dependent decrease in food ingestion and the rate of assimilation to the body upon UV radiation. • Expression of base excision repair-associated and hsp chaperoning genes was significantly increased upon UV radiation in P. nana. - Abstract: To evaluate the effects of ultraviolet (UV) radiation on energy acquisition and consumption, the copepod Paracyclopina nana was irradiated with several doses (0–3 kJ/m{sup 2}) of UV. After UV radiation, we measured the re-brooding success, growth pattern of newly hatched nauplii, ingestion rate, and assimilation of diet. In addition, we checked the modulated patterns of DNA repair and heat shock protein (hsp) chaperoning genes of P. nana. UV-B radiation induced a significant reduction (7–87%) of the re-brooding rate of ovigerous females, indicating that UV-induced egg sac damage is closely correlated with a reduction in the hatching rate of UV-irradiated ovigerous female offspring. Using chlorophyll a and stable carbon isotope incubation experiments, we found a dose-dependent decrease (P < 0.05) in food ingestion and the rate of assimilation to the body in response to UV radiation, implying that P. nana has an underlying ability to shift its balanced-energy status from growth and reproduction to DNA repair and adaptation. Also, expression of P. nana base excision repair (BER)-associated genes and hsp chaperoning genes was significantly increased in response to UV radiation in P. nana. These findings indicate that even 1 kJ/m{sup 2} of UV radiation induces a reduction in reproduction and growth patterns, alters the physiological balance and inhibits the ability to cope with UV-induced damage in P. nana.

  1. In vivo effects of UV radiation on multiple endpoints and expression profiles of DNA repair and heat shock protein (Hsp) genes in the cycloid copepod Paracyclopina nana

    International Nuclear Information System (INIS)

    Won, Eun-Ji; Han, Jeonghoon; Lee, Yeonjung; Kumar, K. Suresh; Shin, Kyung-Hoon; Lee, Su-Jae; Park, Heum Gi; Lee, Jae-Seong

    2015-01-01

    Highlights: • UV-B radiation induced a significant reduction of the re-brooding rate of ovigerous females. • A dose-dependent decrease in food ingestion and the rate of assimilation to the body upon UV radiation. • Expression of base excision repair-associated and hsp chaperoning genes was significantly increased upon UV radiation in P. nana. - Abstract: To evaluate the effects of ultraviolet (UV) radiation on energy acquisition and consumption, the copepod Paracyclopina nana was irradiated with several doses (0–3 kJ/m 2 ) of UV. After UV radiation, we measured the re-brooding success, growth pattern of newly hatched nauplii, ingestion rate, and assimilation of diet. In addition, we checked the modulated patterns of DNA repair and heat shock protein (hsp) chaperoning genes of P. nana. UV-B radiation induced a significant reduction (7–87%) of the re-brooding rate of ovigerous females, indicating that UV-induced egg sac damage is closely correlated with a reduction in the hatching rate of UV-irradiated ovigerous female offspring. Using chlorophyll a and stable carbon isotope incubation experiments, we found a dose-dependent decrease (P < 0.05) in food ingestion and the rate of assimilation to the body in response to UV radiation, implying that P. nana has an underlying ability to shift its balanced-energy status from growth and reproduction to DNA repair and adaptation. Also, expression of P. nana base excision repair (BER)-associated genes and hsp chaperoning genes was significantly increased in response to UV radiation in P. nana. These findings indicate that even 1 kJ/m 2 of UV radiation induces a reduction in reproduction and growth patterns, alters the physiological balance and inhibits the ability to cope with UV-induced damage in P. nana

  2. A vigilant, hypoxia-regulated heme oxygenase-1 gene vector in the heart limits cardiac injury after ischemia-reperfusion in vivo.

    Science.gov (United States)

    Tang, Yao Liang; Qian, Keping; Zhang, Y Clare; Shen, Leping; Phillips, M Ian

    2005-12-01

    The effect of a cardiac specific, hypoxia-regulated, human heme oxygenase-1 (hHO-1) vector to provide cardioprotection from ischemia-reperfusion injury was assessed. When myocardial ischemia and reperfusion is asymptomatic, the damaging effects are cumulative and patients miss timely treatment. A gene therapy approach that expresses therapeutic genes only when ischemia is experienced is a desirable strategy. We have developed a cardiac-specific, hypoxia-regulated gene therapy "vigilant vector'' system that amplifies cardioprotective gene expression. Vigilant hHO-1 plasmids, LacZ plasmids, or saline (n = 40 per group) were injected into mouse heart 2 days in advance of ischemia-reperfusion injury. Animals were exposed to 60 minutes of ischemia followed by 24 hours of reperfusion. For that term (24 hours) effects, the protein levels of HO-1, inflammatory responses, apoptosis, and infarct size were determined. For long-term (3 week) effects, the left ventricular remodeling and recovery of cardiac function were assessed. Ischemia-reperfusion resulted in a timely overexpression of HO-1 protein. Infarct size at 24 hours after ischemia-reperfusion was significantly reduced in the HO-1-treated animals compared with the LacZ-treated group or saline-treated group (P < .001). The reduction of infarct size was accompanied by a decrease in lipid peroxidant activity, inflammatory cell infiltration, and proapoptotic protein level in ischemia-reperfusion-injured myocardium. The long-term study demonstrated that timely, hypoxia-induced HO-1 overexpression is beneficial in conserving cardiac function and attenuating left ventricle remodelling. The vigilant HO-1 vector provides a protective therapy in the heart for reducing cellular damage during ischemia-reperfusion injury and preserving heart function.

  3. Analysis of the Kinetics and Regulation of Cytokine Gene Expression During the Primary In Vivo Immune Response to Killed Brucella Abortus

    Science.gov (United States)

    1992-08-10

    Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in vitro human T cells with...day after immunization viii 47 49 LIST OF TABLES I. PCR primers and Southern blot probes of Th 11Th2 cytokines II. Cytokine mRNA levels in Thyl...sensitive quantitative reverse transcriptase-polymerase chain reaction (RT- PCR ) assay to measure changes in Thl and Th2 cytokine gene expression during

  4. Structurally related antitumor effects of flavanones in vitro and in vivo: involvement of caspase 3 activation, p21 gene expression, and reactive oxygen species production

    International Nuclear Information System (INIS)

    Shen, S.-C.; Ko, C.H.; Tseng, S.-W.; Tsai, S.-H.; Chen, Y.-C.

    2004-01-01

    appearance of DNA ladders, caspase 3 protein processing, PARP protein cleavage, and increasing p21 protein. These results revealed in vitro, ex vivo, and in vivo antitumor activities of 2'-OH flavanone via apoptosis induction, and indicates that 2'-OH flavanone is an active compound worthy of development for cancer chemotherapy

  5. Lipopolysaccharide (LPS) stimulates adipokine and socs3 gene expression in mouse brain and pituitary gland in vivo, and in N-1 hypothalamic neurons in vitro.

    Science.gov (United States)

    Brown, Russell; Imran, Syed A; Wilkinson, Michael

    2009-04-30

    Adipokines that modulate metabolic and inflammatory responses, such as resistin (rstn) and fasting-induced adipose factor (fiaf), are also expressed in mouse brain and pituitary gland. Since lipopolysaccharide (LPS)-induced endotoxinemia provokes an anorectic response via a hypothalamic-dependent mechanism we hypothesized that LPS would also modify hypothalamic adipokine expression. Challenging male CD-1 mice with LPS (5 mg/kg; s.c.) significantly reduced bodyweight (24 h) and realtime RT-PCR revealed time- and tissue-dependent increases in rstn, fiaf and suppressor of cytokine signaling-3 (socs-3) mRNA in hypothalamic, pituitary, cortical and adipose tissues. Gene expression was rapidly increased (3-6 h) in the hypothalamus and pituitary, but returned to normal within 24 h. In contrast, with the exception of rstn in fat, the expression of target genes remained elevated in cortex and visceral fat at 24 h post-injection. In order to more specifically examine the hypothalamic response to LPS we investigated its effects directly on N-1 hypothalamic neurons in vitro. LPS (25 microg/mL; 3 h) had no effect on rstn mRNA, but significantly stimulated fiaf and socs-3 expression. Although various toll-like receptor 4 (TLR4) antagonists (parthenolide, PD098059, and SB202190) did not prevent the LPS-induced increases in fiaf and socs-3, they did partially attenuate its stimulatory effects. We conclude that LPS treatment increases the expression of central, and possibly neuronal, adipokine genes which may influence local tissue repair and function, but could also have downstream consequences on the hypothalamic control of appetite and energy metabolism following an inflammatory insult.

  6. Electroporation-mediated in vivo gene delivery of the Na+/K+-ATPase pump reduced lung injury in a mouse model of lung contusion.

    Science.gov (United States)

    Machado-Aranda, David A; Suresh, M V; Yu, Bi; Raghavendran, Krishnan

    2012-01-01

    Lung contusion (LC) is an independent risk factor for acute respiratory distress syndrome. The final common pathway in ARDS involves accumulation of fluid in the alveoli. In this study, we demonstrate the application of a potential gene therapy approach by delivering the Na+/K+-ATPase pump subunits in a murine model of LC. We hypothesized that restoring the activity of the pump will result in removal of excess alveolar fluid and additionally reduce inflammation. Under anesthesia, C57/BL6 mice were struck along the right posterior axillary line 1 cm above the costal margin with a cortical contusion impactor. Immediately afterward, 100 μg of plasmid DNA coding for the α,β of the Na+/K+-ATPase pump were instilled into the lungs (LC-electroporation-pump group). Contusion only (LC-only) and a sham saline instillation group after contusion were used as controls (LC-electroporation-sham). By using a BTX 830 electroporator, eight electrical pulses of 200 V/cm field strength were applied transthoracically. Mice were killed at 24 hours, 48 hours, and 72 hours after delivery. Bronchial alveolar lavage was recollected to measure albumin and cytokines by enzyme-linked immunosorbent assay. Pulmonary compliance was measured, and lungs were subject to histopathologic analysis. After the electroporation and delivery of genes coding for the α,β subunits of the Na+/K+-ATPase pump, there was a significant mitigation of acute lung injury as evidenced by reduction in bronchial alveolar lavage levels of albumin, improved pressure volume curves, and reduced inflammation seen on histology. Electroporation-mediated gene transfer of the subunits of the Na+/K+-ATPase pump enhanced recovery from acute inflammatory lung injury after LC.

  7. The impacts of neutralized acid mine drainage contaminated water on the expression of selected endocrine-linked genes in juvenile Mozambique tilapia Oreochromis mossambicus exposed in vivo.

    Science.gov (United States)

    Truter, Johannes Christoff; va Wyk, Johannes Hendrik; Oberholster, Paul Johan; Botha, Anna-Maria

    2014-02-01

    Acid mine drainage (AMD) is a global environmental concern due to detrimental impacts on river ecosystems. Little is however known regarding the biological impacts of neutralized AMD on aquatic vertebrates despite excessive discharge into watercourses. The aim of this investigation was to evaluate the endocrine modulatory potential of neutralized AMD, using molecular biomarkers in the teleost fish Oreochromis mossambicus in exposure studies. Surface water was collected from six locations downstream of a high density sludge (HDS) AMD treatment plant and a reference site unimpacted by AMD. The concentrations of 28 elements, including 22 metals, were quantified in the exposure water in order to identify potential links to altered gene expression. Relatively high concentrations of manganese (~ 10mg/l), nickel (~ 0.1mg/l) and cobalt (~ 0.03 mg/l) were detected downstream of the HDS plant. The expression of thyroid receptor-α (trα), trβ, androgen receptor-1 (ar1), ar2, glucocorticoid receptor-1 (gr1), gr2, mineralocorticoid receptor (mr) and aromatase (cyp19a1b) was quantified in juvenile fish after 48 h exposure. Slight but significant changes were observed in the expression of gr1 and mr in fish exposed to water collected directly downstream of the HDS plant, consisting of approximately 95 percent neutralized AMD. The most pronounced alterations in gene expression (i.e. trα, trβ, gr1, gr2, ar1 and mr) was associated with water collected further downstream at a location with no other apparent contamination vectors apart from the neutralized AMD. The altered gene expression associated with the "downstream" locality coincided with higher concentrations of certain metals relative to the locality adjacent to the HDS plant which may indicate a causative link. The current study provides evidence of endocrine disruptive activity associated with neutralized AMD contamination in regard to alterations in the expression of key genes linked to the thyroid, interrenal and

  8. Topical stabilized retinol treatment induces the expression of HAS genes and HA production in human skin in vitro and in vivo.

    Science.gov (United States)

    Li, Wen-Hwa; Wong, Heng-Kuan; Serrano, José; Randhawa, Manpreet; Kaur, Simarna; Southall, Michael D; Parsa, Ramine

    2017-05-01

    Skin Aging manifests primarily with wrinkles, dyspigmentations, texture changes, and loss of elasticity. During the skin aging process, there is a loss of moisture and elasticity in skin resulting in loss of firmness finally leading to skin sagging. The key molecule involved in skin moisture is hyaluronic acid (HA), which has a significant water-binding capacity. HA levels in skin decline with age resulting in decrease in skin moisture, which may contribute to loss of firmness. Clinical trials have shown that topically applied ROL effectively reduces wrinkles and helps retain youthful appearance. In the current study, ROL was shown to induce HA production and stimulates the gene expression of all three forms of hyaluronic acid synthases (HAS) in normal human epidermal keratinocytes monolayer cultures. Moreover, in human skin equivalent tissues and in human skin explants, topical treatment of tissues with a stabilized-ROL formulation significantly induced the gene expression of HAS mRNA concomitant with an increased HA production. Finally, in a vehicle-controlled human clinical study, histochemical analysis confirmed increased HA accumulation in the epidermis in ROL-treated human skin as compared to vehicle. These results show that ROL increases skin expression of HA, a significant contributing factor responsible for wrinkle formation and skin moisture, which decrease during aging. Taken together with the activity to increase collagen, elastin, and cell proliferation, these studies establish that retinol provides multi-functional activity for photodamaged skin.

  9. Short and long term in vivo effects of Cyclosporine A and Sirolimus on genes and proteins involved in lipid metabolism in Wistar rats.

    Science.gov (United States)

    Lopes, Patrícia C; Fuhrmann, Amelia; Sereno, José; Espinoza, Daniel O; Pereira, Maria João; Eriksson, Jan W; Reis, Flávio; Carvalho, Eugenia

    2014-05-01

    Cyclosporine A (CsA) and sirolimus (SRL) are immunosuppressive agents (IA) associated with new onset diabetes after transplantation and dyslipidemia. We aim to evaluate the molecular effects of CsA (5mg/kg/day) and SRL (1mg/kg/day) treatment for 3 and 9weeks on lipid metabolism, in Wistar rats. Lipolysis was evaluated in isolated adipocytes, while triglycerides (TG) and non-esterified fatty acid (NEFA) were measured in serum. Gene and protein expression involved in lipid metabolism was assessed in adipose tissue and liver. CsA and SRL treatments of rats for 3 and 9weeks increased isoproterenol-stimulated lipolysis by 5-9 fold and 4-6 fold in isolated adipocytes, respectively. While CsA increased adipocyte weight and diameter, as well as NEFA and TG levels in circulation after 9weeks, SRL treatment caused ectopic deposition of TG in the liver after 3weeks. Moreover, ACC1 and FAS protein expression was increased after 3weeks (>100%, p42%, pIAs on expression of lipolytic and lipogenic genes suggest that these agents influence lipid metabolism, thus contributing to the dyslipidemia observed during immunosuppressive therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. [Genomic research of traditional Chinese medicines in vivo metabolism].

    Science.gov (United States)

    Xiao, Shui-Ming; Bai, Rui; Zhang, Xiao-Yan

    2016-11-01

    Gene is the base of in vivo metabolism and effectiveness for traditional Chinese medicines (TCM), and the gene expression, regulation and modification are used as the research directions to perform the TCM multi-component, multi-link and multi-target in vivo metabolism studies, which will improve the research on TCM metabolic proecess, effect target and molecular mechanism. Humans are superorganisms with 1% genes inherited from parents and 99% genes from various parts of the human body, mainly coming from the microorganisms in intestinal flora. These indicate that genetically inherited human genome and "second genome" could affect the TCM in vivo metabolism from inheritance and "environmental" aspects respectively. In the present paper, typical case study was used to discuss related TCM in vivo metabolic genomics research, mainly including TCM genomics research and gut metagenomics research, as well as the personalized medicine evoked from the individual difference of above genomics (metagenomics). Copyright© by the Chinese Pharmaceutical Association.

  11. Cadmium analysis in vivo

    International Nuclear Information System (INIS)

    Bergman, R.

    1980-12-01

    The report describes the development of a method for in vivo Cd-analysis. The method is based on the analysis of the prompt gamma radiation which is emitted by neutron capture of the isotope Cd113. Different parts of the body can be analysed selectively by neutrons in the interval of 1 to 100 KeV. The results show that the level of Cd in Kidneys can be measured without exceeding the dose of 40 mrad and that only 20% uncertainty is introduced when analysing Cd. The development has been made at the R2 reactor in Studsvik using 25 KeV neutrons. (G.B.)

  12. Construction of engineering adipose-like tissue in vivo utilizing human insulin gene-modified umbilical cord mesenchymal stromal cells with silk fibroin 3D scaffolds.

    Science.gov (United States)

    Li, Shi-Long; Liu, Yi; Hui, Ling

    2015-12-01

    We evaluated the use of a combination of human insulin gene-modified umbilical cord mesenchymal stromal cells (hUMSCs) with silk fibroin 3D scaffolds for adipose tissue engineering. In this study hUMSCs were isolated and cultured. HUMSCs infected with Ade-insulin-EGFP were seeded in fibroin 3D scaffolds with uniform 50-60 µm pore size. Silk fibroin scaffolds with untransfected hUMSCs were used as control. They were cultured for 4 days in adipogenic medium and transplanted under the dorsal skins of female Wistar rats after the hUMSCs had been labelled with chloromethylbenzamido-1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (CM-Dil). Macroscopical impression, fluorescence observation, histology and SEM were used for assessment after transplantation at 8 and 12 weeks. Macroscopically, newly formed adipose tissue was observed in the experimental group and control group after 8 and 12 weeks. Fluorescence observation supported that the formed adipose tissue originated from seeded hUMSCs rather than from possible infiltrating perivascular tissue. Oil red O staining of newly formed tissue showed that there was substantially more tissue regeneration in the experimental group than in the control group. SEM showed that experimental group cells had more fat-like cells, whose volume was larger than that of the control group, and degradation of the silk fibroin scaffold was greater under SEM observation. This study provides significant evidence that hUMSCs transfected by adenovirus vector have good compatibility with silk fibroin scaffold, and adenoviral transfection of the human insulin gene can be used for the construction of tissue-engineered adipose. Copyright © 2013 John Wiley & Sons, Ltd.

  13. EDITORIAL: Nanotechnology in vivo Nanotechnology in vivo

    Science.gov (United States)

    Demming, Anna

    2010-04-01

    -imaging labels [4]. A surface hydroxyl group renders silicon quantum dots soluble in water and the photoluminescence can be made stable with oxygen-passivation. In addition, researchers in Japan have demonstrated how the initially modest yield in the preparation of silicon quantum dots can be improved to tens of milligrams per batch, thus further promoting their application in bio-imaging [5]. In the search for non-toxic quantum dots, researchers at the Amrita Centre for Nanoscience in India have prepared heavy metal-free quantum dot bio-probes based on single phase ZnS [6]. The quantum dots are selectively doped with metals, transition metals and halides to provide tuneable luminescence properties, and they are surface conjugated with folic acid for cancer targeting. The quantum dots were demonstrated to be water-soluble, non-toxic in normal and cancer cell lines, and have bright, tuneable luminescence. So far most of the quantum dots developed for bio-imaging have had excitation and emission wavelengths in the visible spectrum, which is highly absorbed by tissue. This limits imaging with these quantum dots to superficial tissues. This week, researchers in China and the US reported work developing functionalized dots for in vivo tumour vasculature in the infrared part of the spectrum [7]. In addition the quantum dots were functionalised with glycine-aspartic acid (RGD) peptides, which target the vasculature of almost all types of growing tumours, unlike antibody- or aptamer-mediated targeting strategies that are specific to a particular cancer type. In this issue, researchers in China and the US demonstrate a novel type of contrast agent for ultrasonic tumour imaging [8]. Contrast-enhanced ultrasonic tumour imaging extends the diagnostic and imaging capabilities of traditional techniques. The use of nanoparticles as ultrasound contrast agents exploits the presence of open pores in the range of 380 to 780 nm in tumour blood vessels, which enhance the permeability and retention

  14. Influence of interleukin-1 beta gene polymorphisms on the risk of myocardial infarction and ischemic stroke at young age in vivo and in vitro.

    Science.gov (United States)

    Yang, Bo; Zhao, Hua; X, Bin; Wang, Ya-Bin; Zhang, Jian; Cao, Yu-Kang; Wu, Qing; Cao, Feng

    2015-01-01

    In this study, by using vivo and vitro model, we assessed whether interleukin (IL)-1beta gene polymorphisms influence on the risk of myocardial infarction and ischemic stroke at young age. 147 patients (age stroke were deeded as control group and greed to give blood samples for DNA analysis and biochemical measurements by written informed consent. IL-1β-511 wild type (WT, CC) and SNP (TT) were established and transfected into Rat myocardial H9c2 cell and Mouse brain endothelial bEND.3 cells. In Young Age MI or stroke patients, the IL-1β levels of patients with 511CC are higher than that of patients with 511TT. In our study, NF-κB miRNA, iNOS activity, NF-κB, iNOS and Bax protein expressions of MI-induced H9c2 cell or stroke-induced bEND.3 cells in IL-1β-511TT group were lower than those of IL-1β-511CC. Additionally, the protein expression of MMP-2 of MI-induced H9c2 cell or stroke-induced bEND.3 cells in IL-1β-511TT group were higher than that of IL-1β 511CC group. In conclusion, our data indicate that IL-1β-511TT/CC influence on the risk of myocardial infarction and ischemic stroke at young age through NF-κB, iNOS, MMP-2 and Bax.

  15. Inactivation of the Autolysis-Related Genes lrgB and yycI in Staphylococcus aureus Increases Cell Lysis-Dependent eDNA Release and Enhances Biofilm Development In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Cristiana Ossaille Beltrame

    Full Text Available Staphylococcus aureus ica-independent biofilms are multifactorial in nature, and various bacterial proteins have been associated with biofilm development, including fibronectin-binding proteins A and B, protein A, surface protein SasG, proteases, and some autolysins. The role of extracellular DNA (eDNA has also been demonstrated in some S. aureus biofilms. Here, we constructed a Tn551 library, and the screening identified two genes that affected biofilm formation, lrgB and yycI. The repressive effect of both genes on the development of biofilm was also confirmed in knockout strains constructed by allelic recombination. In contrast, the superexpression of either lrgB or yycI by a cadmium-inducible promoter led to a decrease in biofilm accumulation. Indeed, a significant increase in the cell-lysis dependent eDNA release was detected when lrgB or yycI were inactivated, explaining the enhanced biofilm formed by these mutants. In fact, lrgB and yycI genes belong to distinct operons that repress bacterial autolysis through very different mechanisms. LrgB is associated with the synthesis of phage holin/anti-holin analogues, while YycI participates in the activation/repression of the two-component system YycGF (WalKR. Our in vivo data suggest that autolysins activation lead to increased bacterial virulence in the foreign body animal model since a higher number of attached cells was recovered from the implanted catheters inoculated with lrgB or yycI knockout mutants.

  16. Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo.

    Directory of Open Access Journals (Sweden)

    Suneeta Chimalapati

    Full Text Available Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt, deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection.

  17. Graded Elevation of c-Jun in Schwann Cells In Vivo: Gene Dosage Determines Effects on Development, Remyelination, Tumorigenesis, and Hypomyelination.

    Science.gov (United States)

    Fazal, Shaline V; Gomez-Sanchez, Jose A; Wagstaff, Laura J; Musner, Nicolo; Otto, Georg; Janz, Martin; Mirsky, Rhona; Jessen, Kristján R

    2017-12-13

    Schwann cell c-Jun is implicated in adaptive and maladaptive functions in peripheral nerves. In injured nerves, this transcription factor promotes the repair Schwann cell phenotype and regeneration and promotes Schwann-cell-mediated neurotrophic support in models of peripheral neuropathies. However, c-Jun is associated with tumor formation in some systems, potentially suppresses myelin genes, and has been implicated in demyelinating neuropathies. To clarify these issues and to determine how c-Jun levels determine its function, we have generated c-Jun OE/+ and c-Jun OE/OE mice with graded expression of c-Jun in Schwann cells and examined these lines during development, in adulthood, and after injury using RNA sequencing analysis, quantitative electron microscopic morphometry, Western blotting, and functional tests. Schwann cells are remarkably tolerant of elevated c-Jun because the nerves of c-Jun OE/+ mice, in which c-Jun is elevated ∼6-fold, are normal with the exception of modestly reduced myelin thickness. The stronger elevation of c-Jun in c-Jun OE/OE mice is, however, sufficient to induce significant hypomyelination pathology, implicating c-Jun as a potential player in demyelinating neuropathies. The tumor suppressor P19 ARF is strongly activated in the nerves of these mice and, even in aged c-Jun OE/OE mice, there is no evidence of tumors. This is consistent with the fact that tumors do not form in injured nerves, although they contain proliferating Schwann cells with strikingly elevated c-Jun. Furthermore, in crushed nerves of c-Jun OE/+ mice, where c-Jun levels are overexpressed sufficiently to accelerate axonal regeneration, myelination and function are restored after injury. SIGNIFICANCE STATEMENT In injured and diseased nerves, the transcription factor c-Jun in Schwann cells is elevated and variously implicated in controlling beneficial or adverse functions, including trophic Schwann cell support for neurons, promotion of regeneration, tumorigenesis

  18. Interferon-stimulated gene of 20 kDa protein (ISG20) degrades RNA of hepatitis B virus to impede the replication of HBV in vitro and in vivo

    Science.gov (United States)

    Oshiumi, Hiroyuki; Mengao, Deng; Takaki, Hiromi; Matsumoto, Misako; Aly, Hussein H.; Watashi, Koichi; Chayama, Kazuaki; Seya, Tsukasa

    2016-01-01

    Hepatitis B virus (HBV) barely induces host interferon (IFN)-stimulated genes (ISGs), which allows efficient HBV replication in the immortalized mouse hepatocytes as per human hepatocytes. Here we found that transfection of Isg20 plasmid robustly inhibits the HBV replication in HBV-infected hepatocytes irrespective of IRF3 or IFN promoter activation. Transfection of Isg20 is thus effective to eradicate HBV in the infected hepatocytes. Transfection of HBV genome or ε-stem of HBV pgRNA (active pgRNA moiety) failed to induce Isg20 in the hepatocytes, while control polyI:C (a viral dsRNA analogue mimic) activated MAVS pathway leading to production of type I IFN and then ISGsg20 via the IFN-α/β receptor (IFNAR). Consistently, addition of IFN-α induced Isg20 and partially suppressed HBV replication in hepatocytes. Chasing HBV RNA, DNA and proteins by blotting indicated that ISG20 expression decreased HBV RNA and replicative DNA in HBV-transfected cells, which resulted in low HBs antigen production and virus titer. The exonuclease domains of ISG20 mainly participated in HBV-RNA decay. In vivo hydrodynamic injection, ISG20 was crucial for suppressing HBV replication without degrading host RNA in the liver. Taken together, ISG20 acts as an innate anti-HBV effector that selectively degrades HBV RNA and blocks replication of infectious HBV particles. ISG20 would be a critical effector for ameliorating chronic HBV infection in the IFN therapy. PMID:27626689

  19. An in vivo analysis of Cr6+ induced biochemical, genotoxicological and transcriptional profiling of genes related to oxidative stress, DNA damage and apoptosis in liver of fish, Channa punctatus (Bloch, 1793).

    Science.gov (United States)

    Awasthi, Yashika; Ratn, Arun; Prasad, Rajesh; Kumar, Manoj; Trivedi, Sunil P

    2018-07-01

    Present study was designed to assess the hexavalent chromium (Cr 6+ ) mediated oxidative stress that induces DNA damage and apoptosis in adult fish, Channa punctatus (35 ± 3.0 g; 14.5 ± 1.0 cm; Actinopterygii). Fishes were maintained in three groups for 15, 30 and 45 d of exposure periods. They were treated with 5% (Group T1) and 10% (Group T2) of 96 h-LC 50 of chromium trioxide (Cr 6+ ). Controls were run for the similar duration. A significant (p < 0.05) increment in the activities of antioxidant enzymes, SOD and CAT in liver tissues of the exposed fish evinces the persistence of oxidative stress. A significant (p < 0.05) increase in induction of micronuclei (MN) coupled with transcriptional responses of target genes related to antioxidant enzymes, DNA damage and apoptosis (sod, cat, gsr, nox-1, p53, bax, bcl-2, apaf-1 and casp3a) establishes the impact of oxidative stress due to in vivo, Cr 6+ accumulation in liver as compared to control (0 mg/L), in a dose and exposure-dependent manner. Initially, the increased level of reactive oxygen species (ROS) in liver coincided with that of enhanced mRNA expression of antioxidant enzymes, sod, cat, gsr and nox-1 but, later, the overproduction of ROS, after 45 d of exposure of Cr 6+ , resulted in a significant (p < 0.05) up-regulation of p53. Our findings also unveil that the up-regulation of bax, apaf-1 and casp3a and down-regulation of bcl-2 are associated with Cr 6+ -induced oxidative stress mediated-apoptosis in liver of test fish. Aforesaid molecular markers can, thus, be efficiently utilized for bio-monitoring of aquatic regimes and conservation of fish biodiversity. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Visualization of multidrug resistance in vivo

    International Nuclear Information System (INIS)

    Hendrikse, N.H.; Franssen, E.J.F.; Graaf, W.T.A. van der; Vries, E.G.E. de; Vaalburg, W.

    1999-01-01

    function non-invasively. Results obtained in MRP 2 mutated GY/TR rats have demonstrated visualization of MRP-mediated transport. This tracer permits the study of MRP transport function abnormalities in vivo, e.g. in Dubin-Johnson patients, who are MRP 2 gene deficient. Results obtained show the feasibility of using SPET and PET to study the functionality of MDR transporters in vivo. (orig.)

  1. Herpes simplex virus mutant generation and dual-detection methods for gaining insight into latent/lytic cycles in vivo.

    Science.gov (United States)

    Sawtell, Nancy M; Thompson, Richard L

    2014-01-01

    Two important components to a useful strategy to examine viral gene regulation in vivo are (1) a highly efficient protocol to generate viral mutants that limits undesired mutation and retains full replication competency in vivo and (2) an efficient system to detect and quantify viral promoter activity in rare cells in vivo. Our strategy and protocols for generating, characterizing, and employing HSV viral promoter/reporter mutants in vivo are provided in this two-part chapter.

  2. Nanodiamonds for In Vivo Applications.

    Science.gov (United States)

    van der Laan, KiranJ; Hasani, Masoumeh; Zheng, Tingting; Schirhagl, Romana

    2018-05-01

    Due to their unique optical properties, diamonds are the most valued gemstones. However, beyond the sparkle, diamonds have a number of unique properties. Their extreme hardness gives them outstanding performance as abrasives and cutting tools. Similar to many materials, their nanometer-sized form has yet other unique properties. Nanodiamonds are very inert but still can be functionalized on the surface. Additionally, they can be made in very small sizes and a narrow size distribution. Nanodiamonds can also host very stable fluorescent defects. Since they are protected in the crystal lattice, they never bleach. These defects can also be utilized for nanoscale sensing since they change their optical properties, for example, based on temperature or magnetic fields in their surroundings. In this Review, in vivo applications are focused upon. To this end, how different diamond materials are made and how this affects their properties are discussed first. Next, in vivo biocompatibility studies are reviewed. Finally, the reader is introduced to in vivo applications of diamonds. These include drug delivery, aiding radiology, labeling, and use in cosmetics. The field is critically reviewed and a perspective on future developments is provided. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Transcriptional control by G-quadruplexes: In vivo roles and perspectives for specific intervention.

    Science.gov (United States)

    Armas, Pablo; David, Aldana; Calcaterra, Nora B

    2017-01-01

    G-quadruplexes are non-canonical DNA secondary structures involved in several genomic and molecular processes. Here, we summarize the main G-quadruplex features and evidences proving the in vivo role on the transcriptional regulation of genes required for zebrafish embryonic development. We also discuss alternative strategies for specifically interfering G-quadruplex in vivo.

  4. In vivo evaluation of 2'-deoxy-2'-[{sup 18}F]fluoro-5-iodo-1-{beta}-D-arabinofuranosyluracil ([{sup 18}F]FIAU) and 2'-deoxy-2'-[{sup 18}F]fluoro-5-ethyl-1-{beta}-D-arabinofuranosyluracil ([{sup 18}F]FEAU) as markers for suicide gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Alauddin, Mian M. [University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd. T8.3895, P.O. Box 059, Houston, TX (United States); Shahinian, Antranic; Park, Ryan; Fissekis, John D.; Conti, Peter S. [University of Southern California, PET Imaging Science Center, Keck School of Medicine, Los Angeles, CA (United States); Tohme, Michel [University of Southern California, PET Imaging Science Center, Keck School of Medicine, Los Angeles, CA (United States); Department of Biomedical Engineering, UC Davis, Davis, CA (United States)

    2007-06-15

    FIAU and FEAU were evaluated in vitro and in vivo as markers for HSV1-tk gene expression. In vitro and biodistribution studies were performed in wild type and transduced HT-29 cells using [{sup 14}C]FIAU and [{sup 3}H]FEAU. PET imaging was performed using [{sup 18}F]FIAU and [{sup 18}F]FEAU. In vitro uptake of [{sup 14}C]FIAU in tk-positive cells was 39-fold, 49-fold, and 43-fold higher (p < 0.001) than in wild type cells at 30, 60, and 120 min, respectively. Uptake of [{sup 3}H]FEAU in transduced cells was 46-fold, 62-fold, and 121-fold higher (p < 0.001) than in wild type cells at the same time points. In vivo uptake of [{sup 14}C]FIAU at 2 h in HSV1-tk positive tumors was 15.48 {+-} 3.94, 6.7-fold higher (p < 0.001) than in wild type tumors. Uptake of [{sup 3}H]FEAU in transduced tumors was 9.98 {+-} 1.99, 5.0-fold higher (p < 0.001) than in wild type tumors. Micro-PET images using [{sup 18}F]FIAU and [{sup 18}F]FEAU also showed very high uptake in HSV-tk tumors. [{sup 18}F]FIAU and [{sup 18}F]FEAU appear to be potential PET imaging agents for gene expression. (orig.)

  5. In vivo counting of uranium

    International Nuclear Information System (INIS)

    Palmer, H.E.

    1985-03-01

    A state-of-the-art radiation detector system consisting of six individually mounted intrinsic germanium planar detectors, each 20 cm 2 by 13 mm thick, mounted together such that the angle of the whole system can be changed to match the slope of the chest of the person being counted, is described. The sensitivity of the system for counting uranium and plutonium in vivo and the precedures used in calibrating the system are also described. Some results of counts done on uranium mill workers are presented. 15 figs., 2 tabs

  6. Fatty acid induced changes in gene expression in cultured L6 rat muscle cells : An in vitro model on high dietary fat-induced insulin resistance in red gastrocnemius rat muscle in vivo

    OpenAIRE

    Breivik, Børge

    2004-01-01

    ABSTRACT Type 2 diabetes is a serious cause of morbidity and mortality and the disease is reaching epidemic proportions in the developed world. A core defect in type 2 diabetes is insulin resistance in skeletal muscle. Previous global gene expression experiments conducted at the Garvan Medical Research Institute has shown that 3 weeks high fat feeding induced increased expression of stress related genes in rat muscle. These stress-related genes could be involved in the devel...

  7. Advancing Molecular Therapies through In Vivo Bioluminescent Imaging

    Directory of Open Access Journals (Sweden)

    Anton McCaffrey

    2003-04-01

    Full Text Available Effective development of therapeutics that target the molecular basis of disease is dependent on testing new therapeutic moieties and delivery strategies in animal models of human disease. Accelerating the analyses of these models and improving their predictive value through whole animal imaging methods, which provide data in real time and are sensitive to the subtle changes, are crucial for rapid advancement of these approaches. Modalities based on optics are rapid, sensitive, and accessible methods for in vivo analyses with relatively low instrumentation costs. In vivo bioluminescent imaging (BLI is one of these optically based imaging methods that enable rapid in vivo analyses of a variety of cellular and molecular events with extreme sensitivity. BLI is based on the use of light-emitting enzymes as internal biological light sources that can be detected externally as biological indicators. BLI has been used to test spatio-temporal expression patterns of both target and therapeutic genes in living laboratory animals where the contextual influences of whole biological systems are preserved. BLI has also been used to analyze gene delivery, immune cell therapies, and the in vivo efficacy of inhibitory RNAs. New tools for BLI are being developed that will offer greater flexibility in detection and analyses. BLI can be used to accelerate the evaluation of experimental therapeutic strategies and whole body imaging offers the opportunity of revealing the effects of novel approaches on key steps in disease processes.

  8. Nanomaterials for In Vivo Imaging.

    Science.gov (United States)

    Smith, Bryan Ronain; Gambhir, Sanjiv Sam

    2017-02-08

    In vivo imaging, which enables us to peer deeply within living subjects, is producing tremendous opportunities both for clinical diagnostics and as a research tool. Contrast material is often required to clearly visualize the functional architecture of physiological structures. Recent advances in nanomaterials are becoming pivotal to generate the high-resolution, high-contrast images needed for accurate, precision diagnostics. Nanomaterials are playing major roles in imaging by delivering large imaging payloads, yielding improved sensitivity, multiplexing capacity, and modularity of design. Indeed, for several imaging modalities, nanomaterials are now not simply ancillary contrast entities, but are instead the original and sole source of image signal that make possible the modality's existence. We address the physicochemical makeup/design of nanomaterials through the lens of the physical properties that produce contrast signal for the cognate imaging modality-we stratify nanomaterials on the basis of their (i) magnetic, (ii) optical, (iii) acoustic, and/or (iv) nuclear properties. We evaluate them for their ability to provide relevant information under preclinical and clinical circumstances, their in vivo safety profiles (which are being incorporated into their chemical design), their modularity in being fused to create multimodal nanomaterials (spanning multiple different physical imaging modalities and therapeutic/theranostic capabilities), their key properties, and critically their likelihood to be clinically translated.

  9. In-vivo quantitative measurement

    International Nuclear Information System (INIS)

    Ito, Takashi

    1992-01-01

    So far by positron CT, the quantitative analyses of oxygen consumption rate, blood flow distribution, glucose metabolic rate and so on have been carried out. The largest merit of using the positron CT is the observation and verification of mankind have become easy. Recently, accompanying the rapid development of the mapping tracers for central nervous receptors, the observation of many central nervous receptors by the positron CT has become feasible, and must expectation has been placed on the elucidation of brain functions. The conditions required for in vitro processes cannot be realized in strict sense in vivo. The quantitative measurement of in vivo tracer method is carried out by measuring the accumulation and movement of a tracer after its administration. The movement model of the mapping tracer for central nervous receptors is discussed. The quantitative analysis using a steady movement model, the measurement of dopamine receptors by reference method, the measurement of D 2 receptors using 11C-Racloprode by direct method, and the possibility of measuring dynamics bio-reaction are reported. (K.I.)

  10. The HOX genes are expressed, in vivo, in human tooth germs: in vitro cAMP exposure of dental pulp cells results in parallel HOX network activation and neuronal differentiation.

    Science.gov (United States)

    D'Antò, Vincenzo; Cantile, Monica; D'Armiento, Maria; Schiavo, Giulia; Spagnuolo, Gianrico; Terracciano, Luigi; Vecchione, Raffaela; Cillo, Clemente

    2006-03-01

    Homeobox-containing genes play a crucial role in odontogenesis. After the detection of Dlx and Msx genes in overlapping domains along maxillary and mandibular processes, a homeobox odontogenic code has been proposed to explain the interaction between different homeobox genes during dental lamina patterning. No role has so far been assigned to the Hox gene network in the homeobox odontogenic code due to studies on specific Hox genes and evolutionary considerations. Despite its involvement in early patterning during embryonal development, the HOX gene network, the most repeat-poor regions of the human genome, controls the phenotype identity of adult eukaryotic cells. Here, according to our results, the HOX gene network appears to be active in human tooth germs between 18 and 24 weeks of development. The immunohistochemical localization of specific HOX proteins mostly concerns the epithelial tooth germ compartment. Furthermore, only a few genes of the network are active in embryonal retromolar tissues, as well as in ectomesenchymal dental pulp cells (DPC) grown in vitro from adult human molar. Exposure of DPCs to cAMP induces the expression of from three to nine total HOX genes of the network in parallel with phenotype modifications with traits of neuronal differentiation. Our observations suggest that: (i) by combining its component genes, the HOX gene network determines the phenotype identity of epithelial and ectomesenchymal cells interacting in the generation of human tooth germ; (ii) cAMP treatment activates the HOX network and induces, in parallel, a neuronal-like phenotype in human primary ectomesenchymal dental pulp cells. 2005 Wiley-Liss, Inc.

  11. In vivo fertilization of banana

    Directory of Open Access Journals (Sweden)

    Taliane Leila Soares

    2014-01-01

    Full Text Available The aim of this research was to study the in vivo fertilization process of banana cultivars. The diploid hybrid (AA 091087-01 was the male progenitor. Flower samples were checked for fertilization from the first to the twentieth day after pollination. The size of the diploid ovules increased gradually at the beginning of the seed formation process. On the other hand, in the AAA triploids (Cavendish subgroup, the not fertilized ovules were aborted. In the AAB triploids (Prata subgroup some ovules were fertilized. The flowers of Grand Naine, Nanicão and 'Pacovan' cultivars presented necrosis in the distal part of the ovary on the first day after pollination. Necrosis can hinder pollen tube growth towards the ovule, which might be related to the low seed yield in 'Pacovan' cultivars and to the absence of seeds in the Cavendish subgroup cultivars.

  12. In vitro and in vivo approaches to study osteocyte biology.

    Science.gov (United States)

    Kalajzic, Ivo; Matthews, Brya G; Torreggiani, Elena; Harris, Marie A; Divieti Pajevic, Paola; Harris, Stephen E

    2013-06-01

    Osteocytes, the most abundant cell population of the bone lineage, have been a major focus in the bone research field in recent years. This population of cells that resides within mineralized matrix is now thought to be the mechanosensory cell in bone and plays major roles in the regulation of bone formation and resorption. Studies of osteocytes had been impaired by their location, resulting in numerous attempts to isolate primary osteocytes and to generate cell lines representative of the osteocytic phenotype. Progress has been achieved in recent years by utilizing in vivo genetic technology and generation of osteocyte directed transgenic and gene deficiency mouse models. We will provide an overview of the current in vitro and in vivo models utilized to study osteocyte biology. We discuss generation of osteocyte-like cell lines and isolation of primary osteocytes and summarize studies that have utilized these cellular models to understand the functional role of osteocytes. Approaches that attempt to selectively identify and isolate osteocytes using fluorescent protein reporters driven by regulatory elements of genes that are highly expressed in osteocytes will be discussed. In addition, recent in vivo studies utilizing overexpression or conditional deletion of various genes using dentin matrix protein (Dmp1) directed Cre recombinase are outlined. In conclusion, evaluation of the benefits and deficiencies of currently used cell lines/genetic models in understanding osteocyte biology underlines the current progress in this field. The future efforts will be directed towards developing novel in vitro and in vivo models that would additionally facilitate in understanding the multiple roles of osteocytes. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. The in vitro and in vivo effects of re-expressing methylated von Hippel-Lindau tumor suppressor gene in clear cell renal carcinoma with 5-aza-2'-deoxycytidine.

    Science.gov (United States)

    Alleman, Wade G; Tabios, Ray L; Chandramouli, Gadisetti V R; Aprelikova, Olga N; Torres-Cabala, Carlos; Mendoza, Arnulfo; Rogers, Craig; Rodgers, Craig; Sopko, Nikolai A; Linehan, W Marston; Vasselli, James R

    2004-10-15

    Clear cell renal carcinoma (ccRCC) is strongly associated with loss of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene is functionally lost through hypermethylation in up to 19% of sporadic ccRCC cases. We theorized that re-expressing VHL silenced by methylation in ccRCC cells, using a hypo-methylating agent, may be an approach to treatment in patients with this type of cancer. We test the ability of two hypo-methylating agents to re-express VHL in cell culture and in mice bearing human ccRCC and evaluate the effects of re-expressed VHL in these models. Real-time reverse transcription-PCR was used to evaluate the ability of zebularine and 5-aza-2'-deoxycytidine (5-aza-dCyd) to re-express VHL in four ccRCC cell lines with documented VHL gene silencing through hypermethylation. We evaluated if the VHL re-expressed after hypo-methylating agent treatment could recreate similar phenotypic changes in ccRCC cells observed when the VHL gene is re-expressed via transfection in cell culture and in a xenograft mouse model. Finally we evaluate global gene expression changes occurring in our cells, using microarray analysis. 5-Aza-dCyd was able to re-express VHL in our cell lines both in culture and in xenografted murine tumors. Well described phenotypic changes of VHL expression including decreased invasiveness into Matrigel, and decreased vascular endothelial growth factor and glucose transporter-1 expression were observed in the treated lines. VHL methylated ccRCC xenografted tumors were significantly reduced in size in mice treated with 5-aza-dCyd. Mice bearing nonmethylated but VHL-mutated tumors showed no tumor shrinkage with 5-aza-dCyd treatment. Hypo-methylating agents may be useful in the treatment of patients having ccRCC tumors consisting of cells with methylated VHL.

  14. Structural imprints in vivo decode RNA regulatory mechanisms.

    Science.gov (United States)

    Spitale, Robert C; Flynn, Ryan A; Zhang, Qiangfeng Cliff; Crisalli, Pete; Lee, Byron; Jung, Jong-Wha; Kuchelmeister, Hannes Y; Batista, Pedro J; Torre, Eduardo A; Kool, Eric T; Chang, Howard Y

    2015-03-26

    Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

  15. Absolute calibration in vivo measurement systems

    International Nuclear Information System (INIS)

    Kruchten, D.A.; Hickman, D.P.

    1991-02-01

    Lawrence Livermore National Laboratory (LLNL) is currently investigating a new method for obtaining absolute calibration factors for radiation measurement systems used to measure internally deposited radionuclides in vivo. Absolute calibration of in vivo measurement systems will eliminate the need to generate a series of human surrogate structures (i.e., phantoms) for calibrating in vivo measurement systems. The absolute calibration of in vivo measurement systems utilizes magnetic resonance imaging (MRI) to define physiological structure, size, and composition. The MRI image provides a digitized representation of the physiological structure, which allows for any mathematical distribution of radionuclides within the body. Using Monte Carlo transport codes, the emission spectrum from the body is predicted. The in vivo measurement equipment is calibrated using the Monte Carlo code and adjusting for the intrinsic properties of the detection system. The calibration factors are verified using measurements of existing phantoms and previously obtained measurements of human volunteers. 8 refs

  16. Translating human genetics into mouse: the impact of ultra-rapid in vivo genome editing.

    Science.gov (United States)

    Aida, Tomomi; Imahashi, Risa; Tanaka, Kohichi

    2014-01-01

    Gene-targeted mutant animals, such as knockout or knockin mice, have dramatically improved our understanding of the functions of genes in vivo and the genetic diversity that characterizes health and disease. However, the generation of targeted mice relies on gene targeting in embryonic stem (ES) cells, which is a time-consuming, laborious, and expensive process. The recent groundbreaking development of several genome editing technologies has enabled the targeted alteration of almost any sequence in any cell or organism. These technologies have now been applied to mouse zygotes (in vivo genome editing), thereby providing new avenues for simple, convenient, and ultra-rapid production of knockout or knockin mice without the need for ES cells. Here, we review recent achievements in the production of gene-targeted mice by in vivo genome editing. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  17. Recent molecular approaches to understanding astrocyte function in vivo

    Directory of Open Access Journals (Sweden)

    David eDavila

    2013-12-01

    Full Text Available 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.

  18. In vitro transcription accurately predicts lac repressor phenotype in vivo in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Matthew Almond Sochor

    2014-07-01

    Full Text Available A multitude of studies have looked at the in vivo and in vitro behavior of the lac repressor binding to DNA and effector molecules in order to study transcriptional repression, however these studies are not always reconcilable. Here we use in vitro transcription to directly mimic the in vivo system in order to build a self consistent set of experiments to directly compare in vivo and in vitro genetic repression. A thermodynamic model of the lac repressor binding to operator DNA and effector is used to link DNA occupancy to either normalized in vitro mRNA product or normalized in vivo fluorescence of a regulated gene, YFP. An accurate measurement of repressor, DNA and effector concentrations were made both in vivo and in vitro allowing for direct modeling of the entire thermodynamic equilibrium. In vivo repression profiles are accurately predicted from the given in vitro parameters when molecular crowding is considered. Interestingly, our measured repressor–operator DNA affinity differs significantly from previous in vitro measurements. The literature values are unable to replicate in vivo binding data. We therefore conclude that the repressor-DNA affinity is much weaker than previously thought. This finding would suggest that in vitro techniques that are specifically designed to mimic the in vivo process may be necessary to replicate the native system.

  19. In vivo therapeutic responses contingent on Fanconi anemia/BRCA2 status of the tumor

    NARCIS (Netherlands)

    van der Heijden, Michiel S.; Brody, Jonathan R.; Dezentje, David A.; Gallmeier, Eike; Cunningham, Steven C.; Swartz, Michael J.; DeMarzo, Angelo M.; Offerhaus, G. Johan A.; Isacoff, William H.; Hruban, Ralph H.; Kern, Scott E.

    2005-01-01

    Purpose: BRCA2, FANCC, and FANCG gene mutations are present in a subset of pancreatic cancer. Defects in these genes could lead to hypersensitivity to interstrand cross-linkers in vivo and a more optimal treatment of pancreatic cancer patients based on the genetic profile of the tumor. Experimental

  20. Intrastrain heterogeneity of the mgpB gene in Mycoplasma genitalium is extensive in vitro and in vivo and suggests that variation is generated via recombination with repetitive chromosomal sequences.

    Science.gov (United States)

    Iverson-Cabral, Stefanie L; Astete, Sabina G; Cohen, Craig R; Rocha, Eduardo P C; Totten, Patricia A

    2006-07-01

    Mycoplasma genitalium is associated with reproductive tract disease in women and may persist in the lower genital tract for months, potentially increasing the risk of upper tract infection and transmission to uninfected partners. Despite its exceptionally small genome (580 kb), approximately 4% is composed of repeated elements known as MgPar sequences (MgPa repeats) based on their homology to the mgpB gene that encodes the immunodominant MgPa adhesin protein. The presence of these MgPar sequences, as well as mgpB variability between M. genitalium strains, suggests that mgpB and MgPar sequences recombine to produce variant MgPa proteins. To examine the extent and generation of diversity within single strains of the organism, we examined mgpB variation within M. genitalium strain G-37 and observed sequence heterogeneity that could be explained by recombination between the mgpB expression site and putative donor MgPar sequences. Similarly, we analyzed mgpB sequences from cervical specimens from a persistently infected woman (21 months) and identified 17 different mgpB variants within a single infecting M. genitalium strain, confirming that mgpB heterogeneity occurs over the course of a natural infection. These observations support the hypothesis that recombination occurs between the mgpB gene and MgPar sequences and that the resulting antigenically distinct MgPa variants may contribute to immune evasion and persistence of infection.

  1. In Vivo Imaging of Molecularly Targeted Phage

    Directory of Open Access Journals (Sweden)

    Kimberly A. Kelly

    2006-12-01

    Full Text Available Rapid identification of in vivo affinity ligands would have far-reaching applications for imaging specific molecular targets, in vivo systems imaging, and medical use. We have developed a high-throughput method for identifying and optimizing ligands to map and image biologic targets of interest in vivo. We directly labeled viable phage clones with far-red fluorochromes and comparatively imaged them in vivo by multichannel fluorescence ratio imaging. Using Secreted Protein Acidic and Rich in Cysteine (osteonectin and vascular cell adhesion molecule-1 as model targets, we show that: 1 fluorescently labeled phage retains target specificity on labeling; 2 in vivo distribution can be quantitated (detection thresholds of ~ 300 phage/mm3 tissue throughout the entire depth of the tumor using fluorescent tomographic imaging; and 3 fluorescently labeled phage itself can serve as a replenishable molecular imaging agent. The described method should find widespread application in the rapid in vivo discovery and validation of affinity ligands and, importantly, in the use of fluorochrome-labeled phage clones as in vivo imaging agents.

  2. Anti-tumor effect of adenovirus-mediated suicide gene therapy under control of tumor-specific and radio-inducible chimeric promoter in combination with γ-ray irradiation in vivo

    International Nuclear Information System (INIS)

    Sun Wenjie; Yu Haijun; Xiongjie; Xu Yu; Liao Zhengkai; Zhou Fuxiang; Xie Conghua; Zhou Yunfeng

    2011-01-01

    Objective: To detect the selective inhibitory effects of irradiation plus adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic acid (IAA) suicide gene system using tumor-specific and radio-inducible chimeric promoter on human hepatocellular carcinoma subcutaneously xenografted in nude mouse. Methods: Recombinant replicated-deficient adenovirus vector containing HRP gene and chimeric human telomerase reverse transcriptase (hTERT) promoter carrying 6 radio-inducible CArG elements was constructed. A human subcutaneous transplanting hepatocellular carcinoma (MHCC97 cell line) model was treated with γ-ray irradiation plus intra-tumor injections of adenoviral vector and intra-peritoneal injections of prodrug IAA. The change of tumor volume and tumor growth inhibiting rate, the survival time of nude mice, as well as histopathology of xenograft tumor and normal tissues were evaluated. Results: Thirty one days after the treatment, the relative tumor volumes in the negative, adenovirus therapy, irradiation, and combination groups were 49.23±4.55, 27.71±7.74, 28.53±10.48 and 11.58±3.23, respectively.There was a significantly statistical difference among them (F=16.288, P<0.01).The inhibition effect in the combination group was strongest as compared with that in other groups, and its inhibition ratio was 76.5%. The survival period extended to 43 d in the combination group, which showed a significantly difference with that in the control group (χ 2 =18.307, P<0.01). The area of tumors necrosis in the combination group was larger than that in the other groups, and the normal tissues showed no treatment-related toxic effect in all groups. However, multiple hepatocellular carcinoma metastases were observed in the liver in the control group, there were a few metastases in the monotherapy groups and no metastasis in the combination group. Conclusions: Adenovirus-mediated suicide gene therapy plus radiotherapy dramatically could inhibit tumor growth and prolong

  3. Lymphocyte gene expression signatures from patients and mouse models of hereditary hemochromatosis reveal a function of HFE as a negative regulator of CD8+ T-lymphocyte activation and differentiation in vivo.

    Science.gov (United States)

    Costa, Mónica; Cruz, Eugénia; Oliveira, Susana; Benes, Vladimir; Ivacevic, Tomi; Silva, Maria João; Vieira, Inês; Dias, Francisco; Fonseca, Sónia; Gonçalves, Marta; Lima, Margarida; Leitão, Catarina; Muckenthaler, Martina U; Pinto, Jorge; Porto, Graça

    2015-01-01

    Abnormally low CD8+ T-lymphocyte numbers is characteristic of some patients with hereditary hemochromatosis (HH), a MHC-linked disorder of iron overload. Both environmental and genetic components are known to influence CD8+ T-lymphocyte homeostasis but the role of the HH associated protein HFE is still insufficiently understood. Genome-wide expression profiling was performed in peripheral blood CD8+ T lymphocytes from HH patients selected according to CD8+ T-lymphocyte numbers and from Hfe-/- mice maintained either under normal or high iron diet conditions. In addition, T-lymphocyte apoptosis and cell cycle progression were analyzed by flow cytometry in HH patients. HH patients with low CD8+ T-lymphocyte numbers show a differential expression of genes related to lymphocyte differentiation and maturation namely CCR7, LEF1, ACTN1, NAA50, P2RY8 and FOSL2, whose expression correlates with the relative proportions of naïve, central and effector memory subsets. In addition, expression levels of LEF1 and P2RY8 in memory cells as well as the proportions of CD8+ T cells in G2/M cell cycle phase are significantly different in HH patients compared to controls. Hfe-/- mice do not show alterations in CD8+ T-lymphocyte numbers but differential gene response patterns. We found an increased expression of S100a8 and S100a9 that is most pronounced in high iron diet conditions. Similarly, CD8+ T lymphocytes from HH patients display higher S100a9 expression both at the mRNA and protein level. Altogether, our results support a role for HFE as a negative regulator of CD8+ T-lymphocyte activation. While the activation markers S100a8 and S100a9 are strongly increased in CD8+ T cells from both, Hfe-/- mice and HH patients, a differential profile of genes related to differentiation/maturation of CD8+ T memory cells is evident in HH patients only. This supports the notion that HFE contributes, at least in part, to the generation of low peripheral blood CD8+ T lymphocytes in HH.

  4. A pH and Redox Dual Responsive 4-Arm Poly(ethylene glycol-block-poly(disulfide histamine Copolymer for Non-Viral Gene Transfection in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Kangkang An

    2014-05-01

    Full Text Available A novel 4-arm poly(ethylene glycol-b-poly(disulfide histamine copolymer was synthesized by Michael addition reaction of poly(ethylene glycol (PEG vinyl sulfone and amine-capped poly(disulfide histamine oligomer, being denoted as 4-arm PEG-SSPHIS. This copolymer was able to condense DNA into nanoscale polyplexes (<200 nm in average diameter with almost neutral surface charge (+(5–10 mV. Besides, these polyplexes were colloidal stable within 4 h in HEPES buffer saline at pH 7.4 (physiological environment, but rapidly dissociated to liberate DNA in the presence of 10 mM glutathione (intracellular reducing environment. The polyplexes also revealed pH-responsive surface charges which markedly increased with reducing pH values from 7.4–6.3 (tumor microenvironment. In vitro transfection experiments showed that polyplexes of 4-arm PEG-SSPHIS were capable of exerting enhanced transfection efficacy in MCF-7 and HepG2 cancer cells under acidic conditions (pH 6.3–7.0. Moreover, intravenous administration of the polyplexes to nude mice bearing HepG2-tumor yielded high transgene expression largely in tumor rather other normal organs. Importantly, this copolymer and its polyplexes had low cytotoxicity against the cells in vitro and caused no death of the mice. The results of this study indicate that 4-arm PEG-SSPHIS has high potential as a dual responsive gene delivery vector for cancer gene therapy.

  5. Lymphotoxin prevention of diethylnitrosamine carcinogenesis in vivo

    International Nuclear Information System (INIS)

    Ransom, J.H.; Evans, C.H.; DiPaolo, J.A.

    1982-01-01

    Development of intervention measures to control cancer would be facilitated by being able to monitor in vivo carcinogenesis by in vitro quantitation of early indices of neoplastic transformation to assess the in vivo effectiveness of preventive-therapeutic measures. Pregnant Syrian golden hamsters were used in an in vivo-in vitro transplacental model of carcinogenesis to determine the extent that in vivo administration of immunologic hormone preparations along with chemical carcinogen would prevent morphologic transformation assessed in vitro. Pregnant hamsters at 10-11 days of gestation were given injections ip of 3 mg diethylnitrosamine (DENA)/100 g body weight and were killed 2 days later when fetal cells were seeded for colony formation. The frequency of morphologically transformed colonies was assessed after 7 days of growth. Cloning efficiency and mean transformation frequency after DENA exposure were 3.6% and 1 X 10(-4) per cell seeded, respectively. The ip injection of an immunologic hormone preparation reduced the transformation frequency by 46%. The hormone preparation, containing 10,000 U of lymphotoxin but no detectable interferon, was the ultrafiltered lymphokines (greater than 10,000 mol wt) from phytohemagglutinin-stimulated hamster peritoneal leukocytes. The effect of lymphotoxin on cocarcinogenic exposure of fetal cells to DENA in vivo followed by X-irradiation in vitro was also determined. Cells exposed to 250 rad in vitro had a cloning efficiency of 0.5% and a transformation frequency of 0.4 X 10(-4) per cell seeded. After DENA injection and X-irradiation, the transformation frequency increased to 1 X 10(-4) and was inhibited 64% by lymphotoxin in vivo. Thus immunologic hormones (e.g., lymphotoxin) can prevent carcinogenesis in vivo. Furthermore, in vitro quantitation of transformation is a rapid means for evaluating therapeutic and autochthonous effector mechanisms for their ability to prevent or otherwise modulate carcinogenesis in vivo

  6. Muscle-Driven In Vivo Nanogenerator

    KAUST Repository

    Li, Zhou

    2010-05-05

    (Figure Presented) A nanogenerator based on a single piezoelectric fine wire producing an alternating current (AC) is successfully used for the harvesting of biomechanical energy under in vivo conditions. We demonstrate the implanting and working of such a nanogenerator in a live rat where it harvests energy generated by its breathing or heart beating. This study shows the potential of applying these nanogenerators for driving in vivo nanodevices. © 2010 WILEY-VCH Verlag GmbH & Co. KCaA, Weinheim.

  7. New Genome-Wide Algorithm Identifies Novel In-Vivo Expressed Mycobacterium Tuberculosis Antigens Inducing Human T-Cell Responses with Classical and Unconventional Cytokine Profiles

    DEFF Research Database (Denmark)

    Coppola, Mariateresa; van Meijgaarden, Krista E.; Franken, Kees L. M. C.

    2016-01-01

    -wide transcriptomics of Mtb infected lungs we developed data sets and methods to identify IVE-TB (in-vivo expressed Mtb) antigens expressed in the lung. Quantitative expression analysis of 2,068 Mtb genes from the predicted first operons identified the most upregulated IVE-TB genes during in-vivo pulmonary infection...

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

    Directory of Open Access Journals (Sweden)

    Andrew J Pask

    2008-05-01

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

  9. Studying RNA-protein interactions in vivo by RNA immunoprecipitation

    DEFF Research Database (Denmark)

    Selth, Luke A; Close, Pierre; Svejstrup, Jesper Q

    2011-01-01

    and have significant effects on gene expression. RNA immunoprecipitation (RIP) is a powerful technique used to detect direct and indirect interactions between individual proteins and specific RNA molecules in vivo. Here, we describe RIP methods for both yeast and mammalian cells.......The crucial roles played by RNA-binding proteins in all aspects of RNA metabolism, particularly in the regulation of transcription, have become increasingly evident. Moreover, other factors that do not directly interact with RNA molecules can nevertheless function proximally to RNA polymerases...

  10. Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors

    NARCIS (Netherlands)

    Ahmed, Bushra Y; Chakravarthy, Sridhara; Eggers, Ruben; Hermens, Wim T J M C; Zhang, Jing Ying; Niclou, Simone P; Levelt, Christiaan; Sablitzky, Fred; Anderson, Patrick N; Lieberman, A Robert; Verhaagen, J.

    2004-01-01

    BACKGROUND: Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One

  11. In vivo studies of opiate receptors

    International Nuclear Information System (INIS)

    Frost, J.J.; Dannals, R.F.; Duelfer, T.; Burns, H.D.; Ravert, H.T.; Langstroem, B.; Balasubramanian, V.; Wagner, H.N. Jr.

    1984-01-01

    To study opiate receptors noninvasively in vivo using positron emission tomography, techniques for preferentially labeling opiate receptors in vivo can be used. The rate at which receptor-bound ligand clears from the brain in vivo can be predicted by measuring the equilibrium dissociation constant (KD) at 37 degrees C in the presence of 100 mM sodium chloride and 100 microM guanyl-5'-imidodiphosphate, the drug distribution coefficient, and the molecular weight. A suitable ligand for labeling opiate receptors in vivo is diprenorphine, which binds to mu, delta, and kappa receptors with approximately equal affinity in vitro. However, in vivo diprenorphine may bind predominantly to one opiate receptor subtype, possibly the mu receptor. To predict the affinity for binding to the opiate receptor, a Hansch correlation was determined between the 50% inhibitory concentration for a series of halogen-substituted fentanyl analogs and electronic, lipophilic, and steric parameters. Radiochemical methods for the synthesis of carbon-11-labeled diprenorphine and lofentanil are presented

  12. In vivo studies of opiate receptors

    Energy Technology Data Exchange (ETDEWEB)

    Frost, J.J.; Dannals, R.F.; Duelfer, T.; Burns, H.D.; Ravert, H.T.; Langstroem, B.; Balasubramanian, V.; Wagner, H.N. Jr.

    1984-01-01

    To study opiate receptors noninvasively in vivo using positron emission tomography, techniques for preferentially labeling opiate receptors in vivo can be used. The rate at which receptor-bound ligand clears from the brain in vivo can be predicted by measuring the equilibrium dissociation constant (KD) at 37 degrees C in the presence of 100 mM sodium chloride and 100 microM guanyl-5'-imidodiphosphate, the drug distribution coefficient, and the molecular weight. A suitable ligand for labeling opiate receptors in vivo is diprenorphine, which binds to mu, delta, and kappa receptors with approximately equal affinity in vitro. However, in vivo diprenorphine may bind predominantly to one opiate receptor subtype, possibly the mu receptor. To predict the affinity for binding to the opiate receptor, a Hansch correlation was determined between the 50% inhibitory concentration for a series of halogen-substituted fentanyl analogs and electronic, lipophilic, and steric parameters. Radiochemical methods for the synthesis of carbon-11-labeled diprenorphine and lofentanil are presented.

  13. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  14. Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes

    Directory of Open Access Journals (Sweden)

    Casey A Maguire

    2013-01-01

    Full Text Available Bioluminescence imaging (BLI has shown to be crucial for monitoring in vivo biological processes. So far, only dual bioluminescence imaging using firefly (Fluc and Renilla or Gaussia (Gluc luciferase has been achieved due to the lack of availability of other efficiently expressed luciferases using different substrates. Here, we characterized a codon-optimized luciferase from Vargula hilgendorfii (Vluc as a reporter for mammalian gene expression. We showed that Vluc can be multiplexed with Gluc and Fluc for sequential imaging of three distinct cellular phenomena in the same biological system using vargulin, coelenterazine, and D-luciferin substrates, respectively. We applied this triple imaging system to monitor the effect of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL delivered using an adeno-associated viral vector (AAV on brain tumors in mice. Vluc imaging showed efficient sTRAIL gene delivery to the brain, while Fluc imaging revealed a robust antiglioma therapy. Further, nuclear factor-κB (NF-κB activation in response to sTRAIL binding to glioma cells death receptors was monitored by Gluc imaging. This work is the first demonstration of trimodal in vivo bioluminescence imaging and will have a broad applicability in many different fields including immunology, oncology, virology, and neuroscience.

  15. In vivo cell tracking with bioluminescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung Eun; Kalimuthu, Senthilkumar; Ahn, Byeong Cheol [Dept. of Nuclear Medicine, Kyungpook National University School of Medicine and Hospital, Daegu (Korea, Republic of)

    2015-03-15

    Molecular imaging is a fast growing biomedical research that allows the visual representation, characterization and quantification of biological processes at the cellular and subcellular levels within intact living organisms. In vivo tracking of cells is an indispensable technology for development and optimization of cell therapy for replacement or renewal of damaged or diseased tissue using transplanted cells, often autologous cells. With outstanding advantages of bioluminescence imaging, the imaging approach is most commonly applied for in vivo monitoring of transplanted stem cells or immune cells in order to assess viability of administered cells with therapeutic efficacy in preclinical small animal models. In this review, a general overview of bioluminescence is provided and recent updates of in vivo cell tracking using the bioluminescence signal are discussed.

  16. In Vivo Production of Entomopathogenic Nematodes.

    Science.gov (United States)

    Shapiro-Ilan, David I; Morales-Ramos, Juan A; Rojas, M Guadalupe

    2016-01-01

    In nature, entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects. The nematodes are used widely as biopesticides for suppression of insect pests. More than a dozen entomopathogenic nematode species have been commercialized for use in biological control. Most nematodes intended for commercial application are produced in artificial media via solid or liquid fermentation. However, for laboratory research and small greenhouse or field trials, in vivo production of entomopathogenic nematodes is the common method of propagation. Additionally, small companies continue to produce nematodes using in vivo methods for application in niche markets. Advances in mechanization and alternative production routes (e.g., production geared toward application of nematodes in infected host cadavers) can improve efficiency and economy of scale. The objective of this chapter is to describe basic and advanced procedures for in vivo production of entomopathogenic nematodes.

  17. Parathyroid hormone induces the Nrna family of nuclear orphan receptors in vivo

    International Nuclear Information System (INIS)

    Pirih, Flavia Q.; Aghaloo, Tara L.; Bezouglaia, Olga; Nervina, Jeanne M.; Tetradis, Sotirios

    2005-01-01

    Parathyroid hormone (PTH) has both anabolic and catabolic effects on bone metabolism, although the molecular mechanisms mediating these effects are largely unknown. Among the transcription factors induced by Pth in osteoblasts are the nerve growth factor-inducible factor B (NR4A; NGFI-B) family of orphan nuclear receptors: Nurr1, Nur77, and NOR-1. PTH induces NR4A members through the cAMP-protein kinase A (PKA) pathway in vitro. We report here that PTH rapidly and transiently induced expression of all three NR4A genes in PTH-target tissues in vivo. In calvaria, long bones, and kidneys, NR4A induction was maximal 0.5-1 h after a single intraperitoneal (i.p.) injection of 80 μg/kg PTH. Nur77 demonstrated the highest expression, followed, in order, by Nurr1 and NOR-1. In calvaria and long bone, PTH-induced expression of each NR4A gene was detectable at 10 μg/kg i.p. with maximum induction at 40-80 μg/kg. PTH (3-34) did not induce NR4A mRNA levels in calvaria, long bone, and kidney in vivo, confirming our in vitro results that NR4A genes are induced primarily through the cAMP-PKA pathway. The magnitude of PTH-induced NR4A expression was comparable in vivo and in vitro. However, NR4A mRNA levels peaked and returned to baseline faster in vivo. Both in vivo and in vitro, PTH induced NR4A pre-mRNA levels suggesting that induction of these genes is, at least in part, through activation of mRNA synthesis. The in vivo induction of the NR4A family members by PTH suggests their involvement in, at least some, PTH-induced changes in bone metabolism

  18. In vivo dosimetry in external beam radiotherapy

    International Nuclear Information System (INIS)

    Mijnheer, Ben; Beddar, Sam; Izewska, Joanna; Reft, Chester

    2013-01-01

    In vivo dosimetry (IVD) is in use in external beam radiotherapy (EBRT) to detect major errors, to assess clinically relevant differences between planned and delivered dose, to record dose received by individual patients, and to fulfill legal requirements. After discussing briefly the main characteristics of the most commonly applied IVD systems, the clinical experience of IVD during EBRT will be summarized. Advancement of the traditional aspects of in vivo dosimetry as well as the development of currently available and newly emerging noninterventional technologies are required for large-scale implementation of IVD in EBRT. These new technologies include the development of electronic portal imaging devices for 2D and 3D patient dosimetry during advanced treatment techniques, such as IMRT and VMAT, and the use of IVD in proton and ion radiotherapy by measuring the decay of radiation-induced radionuclides. In the final analysis, we will show in this Vision 20/20 paper that in addition to regulatory compliance and reimbursement issues, the rationale for in vivo measurements is to provide an accurate and independent verification of the overall treatment procedure. It will enable the identification of potential errors in dose calculation, data transfer, dose delivery, patient setup, and changes in patient anatomy. It is the authors’ opinion that all treatments with curative intent should be verified through in vivo dose measurements in combination with pretreatment checks

  19. Computational design of in vivo biomarkers

    International Nuclear Information System (INIS)

    Somogyi, Bálint; Gali, Adam

    2014-01-01

    Fluorescent semiconductor nanocrystals (or quantum dots) are very promising agents for bioimaging applications because their optical properties are superior compared to those of conventional organic dyes. However, not all the properties of these quantum dots suit the stringent criteria of in vivo applications, i.e. their employment in living organisms that might be of importance in therapy and medicine. In our review, we first summarize the properties of an ‘ideal’ biomarker needed for in vivo applications. Despite recent efforts, no such hand-made fluorescent quantum dot exists that may be considered as ‘ideal’ in this respect. We propose that ab initio atomistic simulations with predictive power can be used to design ‘ideal’ in vivo fluorescent semiconductor nanoparticles. We briefly review such ab initio methods that can be applied to calculate the electronic and optical properties of very small nanocrystals, with extra emphasis on density functional theory (DFT) and time-dependent DFT which are the most suitable approaches for the description of these systems. Finally, we present our recent results on this topic where we investigated the applicability of nanodiamonds and silicon carbide nanocrystals for in vivo bioimaging. (topical review)

  20. In vivo X-ray fluorescence analysis

    International Nuclear Information System (INIS)

    Ahlgren, L.

    1980-02-01

    Measurements on five occupationally exposed persons have shown that it is possible to use X-ray fluorescence analysis for in vivo measurements of lead in the skeleton. The technique for calibrating in vivo X-ray fluorescence measurements of lead in bone tissue has been studied in detail and a two-component phantom simulating the bone and the soft tissue parts of the finger constructed. The technique has been used for in vivo measurements on 22 occupationally exposed persons. The minimum detectable concentration of lead in fingerbones was found to be around 20 μg x g -1 . The lead concentrations in their skeletons and blood were compared: the correlation was poor. The variations in lead concentrations in the skeleton have been studied in occupationally exposed persons and in samples from archaeological skeletons. The sensitivity and the minimum detectable concentration of cadmium in the kidney cortex in in vivo measurements has been studied by measurements on kidney models. The minimum detectable concentration was 20 μg x g -1 at a skin-kidney distance of 30 mm and 40 μg x g -1 at 40 mm. Five persons occupationally exposed were studied. (Author)

  1. In-vivo optical investigation of psoriasis

    Science.gov (United States)

    Kapsokalyvas, Dimitrios; Cicchi, Riccardo; Bruscino, Nicola; Alfieri, Domenico; Massi, Daniela; Lotti, Torello; Pavone, Francesco S.

    2011-03-01

    Psoriasis is an autoimmune disease of the skin characterized by hyperkeratosis, hyperproliferation of the epidermis, inflammatory cell accumulation and increased dilatation of dermal papillary blood vessels. Cases of psoriasis were investigated in vivo with optical means in order to evaluate the potential of in vivo optical biopsy. A Polarization Multispectral Dermoscope was employed for the macroscopic observation. Features such as the 'dotted' blood vessels pattern was observed with high contrast. The average size of dot vessels in Psoriasis was measured to be 974 μm2 which is much higher compared to healthy skin. High resolution image sections of the epidermis and the dermis were produced with a custom made Multiphoton Microscope. Imaging extended from the surface of the lesion down to the papillary dermis, at a depth of 200 μm. In the epidermis, a characteristic morphology of the stratum corneum found only in Psoriasis was revealed. Additionally, the cytoplasmic area of the cells in the stratum spinosum layer was found to be smaller than normal. In the dermis the morphological features were more pronounced, where the elongated dermal papillae dominated the papillary layer. Their length exceeds 100μm, which is a far greater value compared to that of healthy skin. These in vivo observations are consistent with the ex vivo histopathological observations, supporting both the applicability and potentiality of multispectral dermoscopy and multiphoton microscopy in the field of in vivo optical investigation and biopsy of skin.

  2. Miniature semiconductor detectors for in vivo dosimetry

    International Nuclear Information System (INIS)

    Rosenfeld, A. B.; Cutajar, D.; Lerch, M. L. F.; Takacs, G.; Cornelius, I. M.; Yudelev, M.; Zaider, M.

    2006-01-01

    Silicon mini-semiconductor detectors are found in wide applications for in vivo personal dosimetry and dosimetry and Micro-dosimetry of different radiation oncology modalities. These applications are based on integral and spectroscopy modes of metal oxide semiconductor field effect transistor and silicon p-n junction detectors. The advantages and limitations of each are discussed. (authors)

  3. In vivo dosimetry in external beam radiotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Mijnheer, Ben [Department of Radiation Oncology, Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Amsterdam 1066 CX (Netherlands); Beddar, Sam [Department of Radiation Physics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030 (United States); Izewska, Joanna [Division of Human Health, International Atomic Energy Agency, Vienna 1400 (Austria); Reft, Chester [Department of Radiation and Cellular Oncology, University of Chicago Medical Center, Chicago, Illinois 60637 (United States)

    2013-07-15

    In vivo dosimetry (IVD) is in use in external beam radiotherapy (EBRT) to detect major errors, to assess clinically relevant differences between planned and delivered dose, to record dose received by individual patients, and to fulfill legal requirements. After discussing briefly the main characteristics of the most commonly applied IVD systems, the clinical experience of IVD during EBRT will be summarized. Advancement of the traditional aspects of in vivo dosimetry as well as the development of currently available and newly emerging noninterventional technologies are required for large-scale implementation of IVD in EBRT. These new technologies include the development of electronic portal imaging devices for 2D and 3D patient dosimetry during advanced treatment techniques, such as IMRT and VMAT, and the use of IVD in proton and ion radiotherapy by measuring the decay of radiation-induced radionuclides. In the final analysis, we will show in this Vision 20/20 paper that in addition to regulatory compliance and reimbursement issues, the rationale for in vivo measurements is to provide an accurate and independent verification of the overall treatment procedure. It will enable the identification of potential errors in dose calculation, data transfer, dose delivery, patient setup, and changes in patient anatomy. It is the authors' opinion that all treatments with curative intent should be verified through in vivo dose measurements in combination with pretreatment checks.

  4. Muscle-Driven In Vivo Nanogenerator

    KAUST Repository

    Li, Zhou; Zhu, Guang; Yang, Rusen; Wang, Aurelia C.; Wang, Zhong Lin

    2010-01-01

    of such a nanogenerator in a live rat where it harvests energy generated by its breathing or heart beating. This study shows the potential of applying these nanogenerators for driving in vivo nanodevices. © 2010 WILEY-VCH Verlag GmbH & Co. KCaA, Weinheim.

  5. 'In vivo' and high resolution spectroscopy in solids by NMR: an instrument for transgenic plants study

    International Nuclear Information System (INIS)

    Colnago, L.A.; Herrmann, P.S.P.; Bernardes Filho, R.

    1995-01-01

    This work has developed a study on transgenic plants using two different techniques of nuclear magnetic resonance, in vivo NMR and high resolution NMR. In order to understand the gene mutations and characterize the plants constituents, NMR spectral data were analysed and discussed, then the results were presented

  6. In vivo gluten challenge in coeliac disease

    Directory of Open Access Journals (Sweden)

    HJ Ellis

    2001-01-01

    Full Text Available In vivo gluten challenge has been used since the early 1950s to study the role of cereal fractions in celiac disease. While early studies relied on crude indicators of celiac toxicity, the advent of jejunal biopsy and sophisticated immunohistochemical techniques has allowed accurate studies to be performed. Studies to determine the nature of the cereal component that is toxic to patients with celiac disease have concentrated on wheat because of its nutritional importance. A number of in vitro studies indicated the presence of one or more celiac-activating epitopes with the N-terminus of the A-gliadin molecule. In vivo challenge with three synthetic peptides subsequently indicated the toxicity of a peptide corresponding to amino acids 31 to 49 of A-gliadin. In vivo gluten challenge is the gold standard for the assessment of celiac toxicity; however, jejunal biopsy is a relatively invasive procedure, thus, other methods have been investigated. Direct infusion of the rectum with gluten has been shown to result in an increase in mucosal intraepithelial lymphocytes, occurring only in celiac patients. This method has been used to study the celiac toxicity of gliadin subfractions. The in vitro technique of small intestinal biopsy organ culture is also a useful tool and appears to give the same results as in vivo challenge. The importance of tiny amounts of gliadin in the diet, such as that which occurs in wheat starch, has been studied by in vivo challenge; this technique has clarified the position of oats in the gluten-free diet. Several studies suggest that this cereal may be included in the diet of most adult celiac patients. Studies of the transport of gliadin across the enterocyte following ingestion or challenge suggest that gliadin may be metabolized by a different pathway in celiac disease. This could result in an abnormal presentation to the immune system, triggering a pathogenic rather than a tolerogenic response.

  7. Microstructural imaging of human neocortex in vivo.

    Science.gov (United States)

    Edwards, Luke J; Kirilina, Evgeniya; Mohammadi, Siawoosh; Weiskopf, Nikolaus

    2018-03-24

    The neocortex of the human brain is the seat of higher brain function. Modern imaging techniques, chief among them magnetic resonance imaging (MRI), allow non-invasive imaging of this important structure. Knowledge of the microstructure of the neocortex has classically come from post-mortem histological studies of human tissue, and extrapolations from invasive animal studies. From these studies, we know that the scale of important neocortical structure spans six orders of magnitude, ranging from the size of axonal diameters (microns), to the size of cortical areas responsible for integrating sensory information (centimetres). MRI presents an opportunity to move beyond classical methods, because MRI is non-invasive and MRI contrast is sensitive to neocortical microstructure over all these length scales. MRI thus allows inferences to be made about neocortical microstructure in vivo, i.e. MRI-based in vivo histology. We review recent literature that has applied and developed MRI-based in vivo histology to probe the microstructure of the human neocortex, focusing specifically on myelin, iron, and neuronal fibre mapping. We find that applications such as cortical parcellation (using R 1 maps as proxies for myelin content) and investigation of cortical iron deposition with age (using R 2 * maps) are already contributing to the frontiers of knowledge in neuroscience. Neuronal fibre mapping in the cortex remains challenging in vivo, but recent improvements in diffusion MRI hold promise for exciting applications in the near future. The literature also suggests that utilising multiple complementary quantitative MRI maps could increase the specificity of inferences about neocortical microstructure relative to contemporary techniques, but that further investment in modelling is required to appropriately combine the maps. In vivo histology of human neocortical microstructure is undergoing rapid development. Future developments will improve its specificity, sensitivity, and

  8. Selection of genetically modified hematopoietic cells in vitro and in vivo using alkylating agent lysomustine.

    Science.gov (United States)

    Rozov, F N; Grinenko, T S; Levit, G L; Krasnov, V P; Belyavsky, A V

    2010-09-15

    Efficient gene transfer into hematopoietic stem cells is vital for the success of gene therapy of hematopoietic and immune system disorders. An in vivo selection system based on a mutant form of the O(6)-methylguanine-DNA-methyltransferase gene (MGMTm) is considered one of the more promising strategies for expansion of hematopoietic cells transduced with viral vectors. Here we demonstrate that MGMTm-expressing cells can be efficiently selected using lysomustine, a nitrosourea derivative of lysine. K562 and murine bone marrow cells expressing MGMTm are protected from the cytotoxic action of lysomustine in vitro. We also show in a murine model that MGMTm-transduced hematopoietic cells can be expanded in vivo on transplantation into sublethally irradiated recipients followed by lysomustine treatment. These results indicate that lysomustine can be used as a potent novel chemoselection drug applicable for gene therapy of hematopoietic and immune system disorders. 2010 Elsevier Inc. All rights reserved.

  9. Duration of in vivo endotoxin tolerance in horses.

    Science.gov (United States)

    Holcombe, Susan J; Jacobs, Carrie C; Cook, Vanessa L; Gandy, Jeffery C; Hauptman, Joseph G; Sordillo, Lorraine M

    2016-05-01

    Endotoxemia models are used to study mechanisms and treatments of early sepsis. Repeated endotoxin exposures induce periods of endotoxin tolerance, characterized by diminished proinflammatory responses to lipopolysaccharide (LPS) and modulated production of proinflammatory cytokines. Repeated measure designs using equine endotoxemia models are rarely performed, despite the advantages associated with reduced variability, because the altered responsiveness would confound study results and because the duration of equine endotoxin tolerance is unknown. We determined the interval of endotoxin tolerance, in vivo, in horses based on physical, clinicopathologic, and proinflammatory gene expression responses to repeated endotoxin exposures. Six horses received 30 ng/kg LPS in saline infused over 30 min. Behavior pain scores, physical examination parameters, and blood for complete blood count and proinflammatory gene expression were obtained at predetermined intervals for 24h. Horses received a total of 3 endotoxin exposures. The first exposure was LPS 1, followed 7 days later by LPS 7 or 14-21 days later by LPS 14-21. Lipopolysaccharide exposures were allocated in a randomized, crossover design. Lipopolysaccharide produced clinical and clinicopathologic signs of endotoxemia and increased expression of tumor necrosis factor alpha (TNFα), interleukin (IL)-6 and IL-8, PHorses exhibited evidence of endotoxin tolerance following LPS 7 but not following LPS 14-21. Horses had significantly lower pain scores, heart rates, respiratory rates and duration of fever, after LPS 7 compared to LPS 1 and LPS 14-21, Phorses after LPS 7, P=0.05. Clinical parameters and TNFα gene expression were similar or slightly increased in horses following LPS 14-21 compared to measurements made in horses following LPS 1, suggesting that endotoxin tolerance had subsided. A minimum of 3 weeks between experiments is warranted if repeated measures designs are used to assess in vivo response to endotoxin in

  10. In Vivo Characterization of a Vertebrate Ultra-conserved Enhancer

    Energy Technology Data Exchange (ETDEWEB)

    Poulin, Francis; Nobrega, Marcelo A.; Plajzer-Frick, Ingrid; Holt, Amy; Afzal, Veena; Rubin, Edward M.; Pennacchio, Len

    2004-10-01

    Genomic sequence comparisons between human, mouse and pufferfish (Takifugu rubripes (Fugu))have revealed a set of extremely conserved noncoding sequences. While this high degree of sequence conservation suggests severe evolutionary constraint and predicts a lack of tolerance to change in order to retain in vivo functionality, such elements have been minimally explored experimentally. In this study, we describe the in-depth characterization of an ancient conserved enhancer, Dc2 located near the dachshund gene, which displays a human-Fugu identity of 84 percent over 424 basepairs (bp). In addition to this large overall conservation, we find that Dc2 is characterized by the presence of a large block of sequence (144 bp) that is completely identical between human, mouse, chicken, zebrafish and Fugu. Through the testing of reporter vector constructs in transgenic mice, we observed that the 424 bp Dc2 conserved element is necessary and sufficient for brain tissue enhancer activity. In vivo analyses also revealed that the 144 bp 100 percent conserved sequence is necessary, but not sufficient, to replicate Dc2 enhancer function. However, the introduction of two separate 16 bp insertions into the highly conserved enhancer core did not cause any detectable modification of its in vivo activity. Our observations indicate that the 144 bp 100 percent conserved element is tolerant of change at least at the resolution of this transgenic mouse assay and suggest that purifying selection on Dc2 sequence might not be as strong as we predicted or that some unknown property also constrains this highly conserved enhancer sequence.

  11. In vivo cellular imaging with microscopes enabled by MEMS scanners

    Science.gov (United States)

    Ra, Hyejun

    High-resolution optical imaging plays an important role in medical diagnosis and biomedical research. Confocal microscopy is a widely used imaging method for obtaining cellular and sub-cellular images of biological tissue in reflectance and fluorescence modes. Its characteristic optical sectioning capability also enables three-dimensional (3-D) image reconstruction. However, its use has mostly been limited to excised tissues due to the requirement of high numerical aperture (NA) lenses for cellular resolution. Microscope miniaturization can enable in vivo imaging to make possible early cancer diagnosis and biological studies in the innate environment. In this dissertation, microscope miniaturization for in vivo cellular imaging is presented. The dual-axes confocal (DAC) architecture overcomes limitations of the conventional single-axis confocal (SAC) architecture to allow for miniaturization with high resolution. A microelectromechanical systems (MEMS) scanner is the central imaging component that is key in miniaturization of the DAC architecture. The design, fabrication, and characterization of the two-dimensional (2-D) MEMS scanner are presented. The gimbaled MEMS scanner is fabricated on a double silicon-on-insulator (SOI) wafer and is actuated by self-aligned vertical electrostatic combdrives. The imaging performance of the MEMS scanner in a DAC configuration is shown in a breadboard microscope setup, where reflectance and fluorescence imaging is demonstrated. Then, the MEMS scanner is integrated into a miniature DAC microscope. The whole imaging system is integrated into a portable unit for research in small animal models of human biology and disease. In vivo 3-D imaging is demonstrated on mouse skin models showing gene transfer and siRNA silencing. The siRNA silencing process is sequentially imaged in one mouse over time.

  12. Epigenetic remodeling and modification to preserve skeletogenesis in vivo.

    Science.gov (United States)

    Godfrey, Tanner C; Wildman, Benjamin J; Javed, Amjad; Lengner, Christopher J; Hassan, Mohammad Quamarul

    2018-12-01

    Current studies offer little insight on how epigenetic remodeling of bone-specific chromatin maintains bone mass in vivo. Understanding this gap and precise mechanism is pivotal for future therapeutic innovation to prevent bone loss. Recently, we found that low bone mass is associated with decreased H3K27 acetylation (activating histone modification) of bone specific gene promoters. Here, we aim to elucidate the epigenetic mechanisms by which a miRNA cluster controls bone synthesis and homeostasis by regulating chromatin accessibility and H3K27 acetylation. In order to decipher the epigenetic axis that regulates osteogenesis, we studied a drug inducible anti-miR-23a cluster (miR-23a Cl ZIP ) knockdown mouse model. MiR-23a cluster knockdown (heterozygous) mice developed high bone mass. These mice displayed increased expression of Runx2 and Baf45a, essential factors for skeletogenesis; and decreased expression of Ezh2, a chromatin repressor indispensable for skeletogenesis. ChIP assays using miR-23a Cl knockdown calvarial cells revealed a BAF45A-EZH2 epigenetic antagonistic mechanism that maintains bone formation. Together, our findings support that the miR-23a Cl connection with tissue-specific RUNX2-BAF45A-EZH2 function is a novel molecular epigenetic axis through which a miRNA cluster orchestrates chromatin modification to elicit major effects on osteogenesis in vivo.

  13. Lack of indirect sensitization in vivo

    International Nuclear Information System (INIS)

    Hetzel, F.W.; Avery, K.; Mensinger, M.; Frinak, S.; Tidwell, C.

    1985-01-01

    The possible utility of respiratory inhibiting drugs as indirect radiation sensitizers has been investigated in both in vitro and in vivo systems. In vitro studies were conducted in V79 monolayer and spheroid cultures examining both respiratory inhibition and radiation survival as end points. These drugs (BCNU, Mustargen and Chlorambucil) were found to be potent respiratory inhibitors and, in the spheroid system, to be effective indirect radiation sensitizers with enhancement ratios of approximately 2.0. In vivo studies with these drugs have also been conducted in a C/sub 3/H mouse MCa tumor system to determine if the same reoxygenation effect observed in spheroids could be demonstrated in the solid tumor system. Detailed microelectrode studies, employing each drug at its LD/sub 10/ level, have been unable to demonstrate any drug induced reoxygenation for any of the drugs tested. Complete details are presented

  14. Advanced tools for in vivo skin analysis.

    Science.gov (United States)

    Cal, Krzysztof; Zakowiecki, Daniel; Stefanowska, Justyna

    2010-05-01

    A thorough examination of the skin is essential for accurate disease diagnostics, evaluation of the effectiveness of topically applied drugs and the assessment of the results of dermatologic surgeries such as skin grafts. Knowledge of skin parameters is also important in the cosmetics industry, where the effects of skin care products are evaluated. Due to significant progress in the electronics and computer industries, sophisticated analytic devices are increasingly available for day-to-day diagnostics. The aim of this article is to review several advanced methods for in vivo skin analysis in humans: magnetic resonance imaging, electron paramagnetic resonance, laser Doppler flowmetry and time domain reflectometry. The molecular bases of these techniques are presented, and several interesting applications in the field are discussed. Methods for in vivo assessment of the biomechanical properties of human skin are also reviewed.

  15. In Vivo Radiobioassay and Research Facility

    International Nuclear Information System (INIS)

    Lynch, Timothy P.

    2011-01-01

    Bioassay monitoring for intakes of radioactive material is an essential part of the internal dosimetry program for radiation workers at the Department of Energy's (DOE) Hanford Site. This monitoring program includes direct measurements of radionuclides in the body by detecting photons that exit the body and analyses of radionuclides in excreta samples. The specialized equipment and instrumentation required to make the direct measurements of these materials in the body are located at the In Vivo Radiobioassay and Research Facility (IVRRF). The IVRRF was originally built in 1960 and was designed expressly for the in vivo measurement of radioactive material in Hanford workers. Most routine in vivo measurements are performed annually and special measurements are performed as needed. The primary source terms at the Hanford Site include fission and activation products (primarily 137Cs and 90Sr), uranium, uranium progeny, and transuranic radionuclides. The facility currently houses five shielded counting systems, men's and women's change rooms and an instrument maintenance and repair shop. Four systems include high purity germanium detectors and one system utilizes large sodium iodide detectors. These systems are used to perform an average of 7,000 measurements annually. This includes approximately 5000 whole body measurements analyzed for fission and activation products and 2000 lung measurements analyzed for americium, uranium, and plutonium. Various other types of measurements are performed periodically to estimate activity in wounds, the thyroid, the liver, and the skeleton. The staff maintains the capability to detect and quantify activity in essentially any tissue or organ. The in vivo monitoring program that utilizes the facility is accredited by the Department of Energy Laboratory Accreditation Program for direct radiobioassay.

  16. Pharmaceutical applications of in vivo EPR

    International Nuclear Information System (INIS)

    Maeder, K.

    1998-01-01

    The aim of this article is to discuss the applications of in vivo EPR in the field of pharmacy. In addition to direct detection of free radical metabolites and measurement of oxygen, EPR can be used to characterize the mechanisms of drug release from biodegradable polymers. Unique information about drug concentration, the microenvironment (viscosity, polarity, pH) and biodistribution (by localized measurement or EPR Imaging) can be obtained. (author)

  17. Adenovirus sequences required for replication in vivo.

    OpenAIRE

    Wang, K; Pearson, G D

    1985-01-01

    We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occup...

  18. - In vivo monitoring of 5-FU

    OpenAIRE

    Lucas, Susanne

    2010-01-01

    The aim of the present study was to establish the in vivo-monitoring of the enrichment of 5-FU liposomes in liver and liver metastases by MRI methods. Relative signal intensities of liver and tumor tissue were determined. After sacrifying animals concentrations of 5-FU and it´s active intracellular metabolite M3 were measured by HPLC techniques. We used CC531 adenocarcinoma in the liver of WAG/Rij rats as a standardized liver tumor model in our investigatio...

  19. In vivo imaging of human biochemistry

    International Nuclear Information System (INIS)

    Hall, L.D.

    1983-01-01

    Positron Emission Tomography (PET) is an extremely powerful method for studying aspects of the biochemistry of defined regions of the human body, literally 'in-vivo' biochemistry. To place this technique in the broader perspective of medical diagnostic methods an introduction is given to some of the more important imaging methods which are already widely used clinically. A brief summary of the most recently developed imaging method, which is based on Nuclear Magnetic Resonance (NMR) Spectroscopy, is also included

  20. Multiphoton spectroscopy of human skin in vivo

    Science.gov (United States)

    Breunig, Hans G.; Weinigel, Martin; König, Karsten

    2012-03-01

    In vivo multiphoton-intensity images and emission spectra of human skin are reported. Optical sections from different depths of the epidermis and dermis have been measured with near-infrared laser-pulse excitation. While the intensity images reveal information on the morphology, the spectra show emission characteristics of main endogenous skin fluorophores like keratin, NAD(P)H, melanin, elastin and collagen as well as of second harmonic generation induced by the excitation-light interaction with the dermal collagen network.

  1. In Vivo Cytogenetic Studies on Aspartame

    OpenAIRE

    AlSuhaibani, Entissar S.

    2010-01-01

    Aspartame (a-Laspartyl-L-phenylalanine 1-methylester) is a dipeptide low-calorie artificial sweetener that is widely used as a nonnutritive sweetener in foods and drinks. The safety of aspartame and its metabolic breakdown products (phenylalanine, aspartic acid and methanol) was investigated in vivo using chromosomal aberration (CA) test and sister chromatid exchange (SCE) test in the bone marrow cells of mice. Swiss Albino male mice were exposed to aspartame (3.5, 35, 350 mg/kg body weight)....

  2. Cells in Dengue Virus Infection In Vivo

    Directory of Open Access Journals (Sweden)

    Sansanee Noisakran

    2010-01-01

    Full Text Available Dengue has been recognized as one of the most important vector-borne emerging infectious diseases globally. Though dengue normally causes a self-limiting infection, some patients may develop a life-threatening illness, dengue hemorrhagic fever (DHF/dengue shock syndrome (DSS. The reason why DHF/DSS occurs in certain individuals is unclear. Studies in the endemic regions suggest that the preexisting antibodies are a risk factor for DHF/DSS. Viremia and thrombocytopenia are the key clinical features of dengue virus infection in patients. The amounts of virus circulating in patients are highly correlated with severe dengue disease, DHF/DSS. Also, the disturbance, mainly a transient depression, of hematological cells is a critical clinical finding in acute dengue patients. However, the cells responsible for the dengue viremia are unresolved in spite of the intensive efforts been made. Dengue virus appears to replicate and proliferate in many adapted cell lines, but these in vitro properties are extremely difficult to be reproduced in primary cells or in vivo. This paper summarizes reports on the permissive cells in vitro and in vivo and suggests a hematological cell lineage for dengue virus infection in vivo, with the hope that a new focus will shed light on further understanding of the complexities of dengue disease.

  3. Gut proteases target Yersinia invasin in vivo

    Directory of Open Access Journals (Sweden)

    Freund Sandra

    2011-04-01

    Full Text Available Abstract Background Yersinia enterocolitica is a common cause of food borne gastrointestinal disease. After oral uptake, yersiniae invade Peyer's patches of the distal ileum. This is accomplished by the binding of the Yersinia invasin to β1 integrins on the apical surface of M cells which overlie follicle associated lymphoid tissue. The gut represents a barrier that severely limits yersiniae from reaching deeper tissues such as Peyer's patches. We wondered if gut protease attack on invasion factors could contribute to the low number of yersiniae invading Peyer's patches. Findings Here we show that invasin is rapidly degraded in vivo by gut proteases in the mouse infection model. In vivo proteolytic degradation is due to proteolysis by several gut proteases such as trypsin, α-chymotrypsin, pancreatic elastase, and pepsin. Protease treated yersiniae are shown to be less invasive in a cell culture model. YadA, another surface adhesin is cleaved by similar concentrations of gut proteases but Myf was not cleaved, showing that not all surface proteins are equally susceptible to degradation by gut proteases. Conclusions We demonstrate that gut proteases target important Yersinia virulence factors such as invasin and YadA in vivo. Since invasin is completely degraded within 2-3 h after reaching the small intestine of mice, it is no longer available to mediate invasion of Peyer's patches.

  4. In vivo dosimetry during breast irradiation

    International Nuclear Information System (INIS)

    Ebert, M.A.; Herbert, C.E.; Joseph, D.J.

    2001-01-01

    Full text: In vivo dosimetry during breast irradiation can be difficult due to frequent use of wedged fields, and the contour of the breast. In vivo measurements of central-axis entrance dose were made on 62 breast patients for two consecutive fractions. Discrepancies from expected doses of up to 13.4 % were found for lateral tangential fields (mean 4.31 %). It was proposed that large discrepancies were due to i) dosimetric I and setup errors, and ii) diode misplacement errors. An investigation of the effect of diode misplacement error was undertaken by reviewing possible measurement errors for 20 randomly selected breast treatments. A Monte Carlo study was used to examine the expected measurement error as a function of the standard deviation (SD) in diode placement error (see figure). A strong relationship was found between breast contour and wedge angle. Diode misplacement in the presence of a large wedge angle was identified as a major possible source of measurement error. For the sample of treatments considered, the Monte Carlo study showed that, ignoring general dosimetric errors, mean errors of 4 % are feasible for setup errors of the order of 1 cm (the width of the diode). This study has shown that accurate in vivo dosimetry requires separating measurements errors out from diode readings in order not to overestimate the actual dosimetric errors occurring at treatment time. Copyright (2001) Australasian College of Physical Scientists and Engineers in Medicine

  5. Efficient in vivo electroporation of the postnatal rodent forebrain.

    Directory of Open Access Journals (Sweden)

    Camille Boutin

    Full Text Available Functional gene analysis in vivo represents still a major challenge in biomedical research. Here we present a new method for the efficient introduction of nucleic acids into the postnatal mouse forebrain. We show that intraventricular injection of DNA followed by electroporation induces strong expression of transgenes in radial glia, neuronal precursors and neurons of the olfactory system. We present two proof-of-principle experiments to validate our approach. First, we show that expression of a human isoform of the neural cell adhesion molecule (hNCAM-140 in radial glia cells induces their differentiation into cells showing a neural precursor phenotype. Second, we demonstrate that p21 acts as a cell cycle inhibitor for postnatal neural stem cells. This approach will represent an important tool for future studies of postnatal neurogenesis and of neural development in general.

  6. Setup of IN VIVO Breast Cancer Models for Nanodrug Testing

    DEFF Research Database (Denmark)

    Schifter, Søren

    2013-01-01

    RNA/aptamer conjugates, or carriers such as liposome/chitosan/micelle spheres. As a first step towards testing of the efficacy of siRNA delivery in vivo via different conjugates and complexes, we aimed at developing a standardized breast cancer model system in mice. In this conception, a reporter gene is used...... differential knockdown efficacies and the readout can directly be performed by quantitative imaging using a Caliper IVIS system. In one line of experiments, we engineered non-metastatic MCF-7 breast cancer cells to express the luminescent reporter firefly luciferase (Luc2) along with a pro-metastatic micro......Synthetic lethality is a promising concept for future cancer treatment and using siRNAs as the synthetic lethal drug component allows for also covering the space considered as non-druggable by conventional small molecule drugs. Systemic administration of naked siRNA, however, does not result...

  7. Low density lipoprotein receptors: preliminary results on 'in vivo' study

    International Nuclear Information System (INIS)

    Lupattelli, G.; Virgolini, I.; Li, S.R.; Sinzinger, H.

    1991-01-01

    Plasmatic levels of low density lipoproteins (LDL) are regulated by the receptor pathway and most LDL receptor are located in the liver. A receptor defect due to genetic mutations of the LDL receptor gene is the cause of familial hypercholesterolemia (F.H.), a disease characterized by high cholesterol levels and premature atherosclerosis. Injections of autologous radiolabelled LDL, followed by hepatic scintiscanning, can be used to obtain 'in vivo' quantification of hepatic receptor activity, both in normal and hypercholesterolemic patients. In this study we observe no hepatic increase of radioactivity in patients affected by F.H., confirming the liver receptor defect. Scintigraphy is a non-invasive technique which can be used to diagnose this disease and to monitor the efficiacy of hypolipidemic therapy. (Authors)

  8. Real-time dynamic imaging of virus distribution in vivo.

    Directory of Open Access Journals (Sweden)

    Sean E Hofherr

    2011-02-01

    Full Text Available The distribution of viruses and gene therapy vectors is difficult to assess in a living organism. For instance, trafficking in murine models can usually only be assessed after sacrificing the animal for tissue sectioning or extraction. These assays are laborious requiring whole animal sectioning to ascertain tissue localization. They also obviate the ability to perform longitudinal or kinetic studies in one animal. To track viruses after systemic infection, we have labeled adenoviruses with a near-infrared (NIR fluorophore and imaged these after intravenous injection in mice. Imaging was able to track and quantitate virus particles entering the jugular vein simultaneous with injection, appearing in the heart within 500 milliseconds, distributing in the bloodstream and throughout the animal within 7 seconds, and that the bulk of virus distribution was essentially complete within 3 minutes. These data provide the first in vivo real-time tracking of the rapid initial events of systemic virus infection.

  9. Disappearance of nucleosome positioning in mitotic chromatin in vivo.

    Science.gov (United States)

    Komura, Jun-ichiro; Ono, Tetsuya

    2005-04-15

    During mitosis, transcription is silenced and most transcription factors are displaced from their recognition sequences. By in vivo footprinting analysis, we have confirmed and extended previous studies showing loss of transcription factors from an RNA polymerase II promoter (c-FOS) and, for the first time, an RNA polymerase III promoter (U6) in HeLa cells. Because little was known about nucleosomal organization in mitotic chromosomes, we performed footprinting analysis for nucleosomes on these promoters in interphase and mitotic cells. During interphase, each of the promoters had a positioned nucleosome in the region intervening between proximal promoter elements and distal enhancer elements, but the strong nucleosome positioning disappeared during mitosis. Thus, the nucleosomal organization that appears to facilitate transcription in interphase cells may be lost in mitotic cells, and nucleosome positioning during mitosis does not seem to be a major component of the epigenetic mechanisms to mark genes for rapid reactivation after this phase.

  10. Construction of radiation - induced metastasis model in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jong Kuk; Jang, Su Jin; Kang, Sung Wook; Kim, Jae Sung; Hwang, Sang Gu; Kang, Joo Hyun [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2011-05-15

    In treatment of cancer, distant metastases are important limiting factor because an estimated 50% of all cancer patients will develop metastases, and the metastases are major causing of cancer treatment failure. Recently a few reports indicated {gamma}-radiation induced an increase of invasiveness of several cancer cells. In this study, we had tried to show the possibility that radiation could also induce metastasis in vivo system. To prove our hypothesis, we constructed primary tumor by using C6-TL transfectant cell line expressing HSV1-tk and firefly luciferase (fLuc), and then {gamma}-radiation was treated to xenografts locally. Treatment of {gamma}-radiation to primary C6-TL xenografts of mice reduced size of xenografts and elongated survival of mice than those of mock control mice. But we also show that {gamma}-radiation treatment was followed by the growth of dormant metastases in various organs including lung and intestine after 2-4 weeks of {gamma}-radiation treatment. When bioluminescence imaging indicated growth of tumor in organs in mice, we sacrificed the mice and repeat acquired bioluminescence imaging after repeatedly. These images presented tumor growth locations exactly in organs. Because metastatic tumor candidates have morphology of foci, biopsies were performed for histological analysis or PCR analysis to confirm metastases. In most foci, histological analysis indicated several features of typical cancer tissue and PCR analysis showed present of fLuc gene in metastases. Detection of fLuc gene in metastases indicated these foci were originated from primary C6-TL xenografts, and the results suggest that {gamma}-radiation could promote metastasis in vivo as well as in vitro system. Although we need to understand changes of intracellular signaling or physiological phenomena of the radiation-induced metastasis yet, these results also imply that {gamma}-radiation treatment only to cancer patients need to pay attention carefully, and development of new

  11. Near-infrared optical imaging of nucleic acid nanocarriers in vivo.

    Science.gov (United States)

    Rome, Claire; Gravier, Julien; Morille, Marie; Divita, Gilles; Bolcato-Bellemin, Anne-Laure; Josserand, Véronique; Coll, Jean-Luc

    2013-01-01

    Noninvasive, real-time optical imaging methods are well suited to follow the in vivo distribution of nucleic acid nanocarriers, their dissociation, and the resulting gene expression or inhibition. Indeed, most small animal imaging devices perform bioluminescence and fluorescence measurements without moving the animal, allowing a simple, rapid, and cost-effective method of investigation of several parameters at a time, in longitudinal experiments that can last for days or weeks.Here we help the reader in choosing adapted near-infrared (NIR) fluorophores or pairs of fluorophores for Förster resonance energy transfer assays, imaging of reporter genes, as well as nanocarriers for in vivo gene and siRNA delivery. In addition, we present the labeling methods of these macromolecules and of their payload and the protocols to detect them using bioluminescence and NIR fluorescence imaging in mice.

  12. In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

    DEFF Research Database (Denmark)

    Hojman, Pernille; Eriksen, Jens; Gehl, Julie

    2009-01-01

    DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly...... weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability....... efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8...

  13. In Vivo RNAi-Based Screens: Studies in Model Organisms

    Directory of Open Access Journals (Sweden)

    Miki Yamamoto-Hino

    2013-11-01

    Full Text Available RNA interference (RNAi is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA and target mRNA molecules. Numerous cell-based genome-wide screens have successfully identified novel genes involved in various biological processes, including signal transduction, cell viability/death, and cell morphology. However, cell-based screens cannot address cellular processes such as development, behavior, and immunity. Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening. Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system. Using this inducible RNAi technique, various large-scale screens have been performed in Drosophila, demonstrating that the method is straightforward and valuable. However, accumulated results reveal that the results of RNAi-based screens have relatively high levels of error, such as false positives and negatives. Here, we review in vivo RNAi screens in Drosophila and the methods that could be used to remove ambiguity from screening results.

  14. Expression of nucleolar-related proteins in porcine preimplantation embryos produced in vivo and in vitro

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Wrenzycki, Christine; Strejcek, Frantisek

    2004-01-01

    The expression of nucleolar-related proteins was studied as an indirect marker of the ribosomal RNA (rRNA) gene activation in porcine embryos up to the blastocyst stage produced in vivo and in vitro. A group of the in vivo-developed embryos were cultured with alpha-amanitin to block the de novo...... proteins pRb and p130, which are involved in cell-cycle regulation, was assessed by semiquantitative RT-PCR up to the blastocyst stage. Toward the end of third cell cycle, the nuclei in non-alpha-amanitin-treated, in vivo-produced embryos displayed different stages of transformation of the nuclear...... was delayed in porcine embryos produced in vitro compared to the in vivo-derived counterparts with respect to mRNAs encoding PAF53 and UBF. Moreover, differences existed in the mRNA expression patterns of pRb between in vivo- and in vitro-developed embryos. These findings show, to our knowledge for the first...

  15. In vivo and in vitro studies of cartilage differentiation in altered gravities

    Science.gov (United States)

    Montufar-Solis, D.; Duke, P. J.; D'Aunno, D.

    The in vivo model our laboratory uses for studies of cartilage differentiation in space is the rat growth plate. Differences between missions, and in rat age and recovery times, provided differing results from each mission. However, in all missions, proliferation and differentiation of chondrocytes in the epiphyseal plate of spaceflown rats was altered as was matrix organization. In vitro systems, necessary complements to in vivo work, provide some advantages over the in vivo situation. In vitro, centrifugation of embryonic limb buds suppressed morphogenesis due to precocious differentiation, and changes in the developmental pattern suggest the involvement of Hox genes. In space, embryonic mouse limb mesenchyme cells differentiating in vitro on IML-1 had smoother membranes and lacked matrix seen in controls. Unusual formations, possibly highly ruffled membranes, were found in flight cultures. These results, coupled with in vivo centrifugation studies, show that in vivo or in vitro, the response of chondrocytes to gravitational changes follows Hert's curve as modified by Simon, i.e. decreased loading decreases differentiation, and increased loading speeds it up, but only to a point. After that, additional increases again slow down chondrogenesis.

  16. In vivo virtual intraoperative surgical photoacoustic microscopy

    International Nuclear Information System (INIS)

    Han, Seunghoon; Kim, Sehui; Kim, Jeehyun; Lee, Changho; Jeon, Mansik; Kim, Chulhong

    2013-01-01

    We developed a virtual intraoperative surgical photoacoustic microscopy system by combining with a commercial surgical microscope and photoacoustic microscope (PAM). By sharing the common optical path in the microscope and PAM system, we could acquire the PAM and microscope images simultaneously. Moreover, by employing a beam projector to back-project 2D PAM images onto the microscope view plane as augmented reality, the conventional microscopic and 2D cross-sectional PAM images are concurrently mapped on the plane via an ocular lens of the microscope in real-time. Further, we guided needle insertion into phantom ex vivo and mice skins in vivo

  17. Audio frequency in vivo optical coherence elastography

    Science.gov (United States)

    Adie, Steven G.; Kennedy, Brendan F.; Armstrong, Julian J.; Alexandrov, Sergey A.; Sampson, David D.

    2009-05-01

    We present a new approach to optical coherence elastography (OCE), which probes the local elastic properties of tissue by using optical coherence tomography to measure the effect of an applied stimulus in the audio frequency range. We describe the approach, based on analysis of the Bessel frequency spectrum of the interferometric signal detected from scatterers undergoing periodic motion in response to an applied stimulus. We present quantitative results of sub-micron excitation at 820 Hz in a layered phantom and the first such measurements in human skin in vivo.

  18. Audio frequency in vivo optical coherence elastography

    International Nuclear Information System (INIS)

    Adie, Steven G; Kennedy, Brendan F; Armstrong, Julian J; Alexandrov, Sergey A; Sampson, David D

    2009-01-01

    We present a new approach to optical coherence elastography (OCE), which probes the local elastic properties of tissue by using optical coherence tomography to measure the effect of an applied stimulus in the audio frequency range. We describe the approach, based on analysis of the Bessel frequency spectrum of the interferometric signal detected from scatterers undergoing periodic motion in response to an applied stimulus. We present quantitative results of sub-micron excitation at 820 Hz in a layered phantom and the first such measurements in human skin in vivo.

  19. In vivo P-31 MR diffusion spectroscopy

    International Nuclear Information System (INIS)

    Moonen, C.T.W.; Vanzijl, P.C.M.; LeBihan, D.

    1988-01-01

    This paper discusses the Stejskal-Tanner diffusion spin-echo sequence modified for the in vivo diffusion spectroscopy. The apparent diffusion constant D α was measured as a function of the diffusion time. Contrary to the results in phantom samples, a strong dependency of the D α for phosphocreatine (PCr) in the rat muscle tissue on diffusion time was observed, clearly indicating restricted diffusion effects and allowing an approximation of the size of the restricted volume (8-13 μm). This size fits well with the known dimensions of a normal muscle cell

  20. In vivo virtual intraoperative surgical photoacoustic microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Han, Seunghoon, E-mail: hsh860504@gmail.com; Kim, Sehui, E-mail: sehui0916@nate.com; Kim, Jeehyun, E-mail: jeehk@knu.ac.kr, E-mail: chulhong@postech.edu [School of Electrical Engineering and Computer Science, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Lee, Changho, E-mail: ch31037@postech.edu; Jeon, Mansik, E-mail: msjeon@postech.edu [Department of Creative IT Engineering, Pohang University of Science and Technology (POSTECH), Pohang 790-784 (Korea, Republic of); Kim, Chulhong, E-mail: jeehk@knu.ac.kr, E-mail: chulhong@postech.edu [Department of Creative IT Engineering, Pohang University of Science and Technology (POSTECH), Pohang 790-784 (Korea, Republic of); Department of Biomedical Engineering, The State University of New York at Buffalo, Buffalo, New York 14221 (United States)

    2013-11-11

    We developed a virtual intraoperative surgical photoacoustic microscopy system by combining with a commercial surgical microscope and photoacoustic microscope (PAM). By sharing the common optical path in the microscope and PAM system, we could acquire the PAM and microscope images simultaneously. Moreover, by employing a beam projector to back-project 2D PAM images onto the microscope view plane as augmented reality, the conventional microscopic and 2D cross-sectional PAM images are concurrently mapped on the plane via an ocular lens of the microscope in real-time. Further, we guided needle insertion into phantom ex vivo and mice skins in vivo.

  1. Sampling system for in vivo ultrasound images

    DEFF Research Database (Denmark)

    Jensen, Jorgen Arendt; Mathorne, Jan

    1991-01-01

    Newly developed algorithms for processing medical ultrasound images use the high frequency sampled transducer signal. This paper describes demands imposed on a sampling system suitable for acquiring such data and gives details about a prototype constructed. It acquires full clinical images...... at a sampling frequency of 20 MHz with a resolution of 12 bits. The prototype can be used for real time image processing. An example of a clinical in vivo image is shown and various aspects of the data acquisition process are discussed....

  2. Transcription of human 7S K DNA in vitro and in vivo is exclusively controlled by an upstream promoter

    Energy Technology Data Exchange (ETDEWEB)

    Kleinert, H.; Benecke, B.J.

    1988-02-25

    The authors have analyzed the transcription of a recently isolated human 7S K RNA gene in vitro and in vivo. In contrast to hitherto characterized class III genes (genes transcribed by RNA polymerase III), the coding sequence of this gene is not required for faithful and efficient transcription by RNA polymerase III. In fact, a procaryotic vector DNA sequence was efficiently transcribed by RNA polymerase III under the control of the 7S K RNA gene upstream sequence in vitro and in vivo. S/sub 1/-nuclease protection analyses confirmed that the 7S K 5'flanking sequence was sufficient for accurate transcription initiation. These data demonstrate that 7S K DNA represents a novel class III gene, the promoter elements of which are located outside the coding sequence.

  3. Development and analysis of an in vivo-compatible metabolic network of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Reifman Jaques

    2010-11-01

    Full Text Available Abstract Background During infection, Mycobacterium tuberculosis confronts a generally hostile and nutrient-poor in vivo host environment. Existing models and analyses of M. tuberculosis metabolic networks are able to reproduce experimentally measured cellular growth rates and identify genes required for growth in a range of different in vitro media. However, these models, under in vitro conditions, do not provide an adequate description of the metabolic processes required by the pathogen to infect and persist in a host. Results To better account for the metabolic activity of M. tuberculosis in the host environment, we developed a set of procedures to systematically modify an existing in vitro metabolic network by enhancing the agreement between calculated and in vivo-measured gene essentiality data. After our modifications, the new in vivo network contained 663 genes, 838 metabolites, and 1,049 reactions and had a significantly increased sensitivity (0.81 in predicted gene essentiality than the in vitro network (0.31. We verified the modifications generated from the purely computational analysis through a review of the literature and found, for example, that, as the analysis suggested, lipids are used as the main source for carbon metabolism and oxygen must be available for the pathogen under in vivo conditions. Moreover, we used the developed in vivo network to predict the effects of double-gene deletions on M. tuberculosis growth in the host environment, explore metabolic adaptations to life in an acidic environment, highlight the importance of different enzymes in the tricarboxylic acid-cycle under different limiting nutrient conditions, investigate the effects of inhibiting multiple reactions, and look at the importance of both aerobic and anaerobic cellular respiration during infection. Conclusions The network modifications we implemented suggest a distinctive set of metabolic conditions and requirements faced by M. tuberculosis during

  4. In vitro and in vivo Development of Cloned Ovine Embryos using in vitro and in vivo Matured Oocytes

    DEFF Research Database (Denmark)

    Holm, P; Nagashima, H; Sun, F-J

    1995-01-01

    Cloning of sheep embryos by nucleus transplantation can be achieved by using in vivo matured (oviductal) oocytes and in vivo culture. However, these steps involve cumbersome procedures. Therefore, the effects of in vivo vs. the equivalent in vitro procedures on the pre-implantation development of...

  5. Endogenous fibrinolysis facilitates clot retraction in vivo.

    Science.gov (United States)

    Samson, Andre L; Alwis, Imala; Maclean, Jessica A A; Priyananda, Pramith; Hawkett, Brian; Schoenwaelder, Simone M; Jackson, Shaun P

    2017-12-07

    Clot retraction refers to the process whereby activated platelets transduce contractile forces onto the fibrin network of a thrombus, which over time increases clot density and decreases clot size. This process is considered important for promoting clot stability and maintaining blood vessel patency. Insights into the mechanisms regulating clot retraction at sites of vascular injury have been hampered by a paucity of in vivo experimental models. By pairing localized vascular injury with thrombin microinjection in the mesenteric circulation of mice, we have demonstrated that the fibrin network of thrombi progressively compacts over a 2-hour period. This was a genuine retraction process, as treating thrombi with blebbistatin to inhibit myosin IIa-mediated platelet contractility prevented shrinkage of the fibrin network. Real-time confocal analysis of fibrinolysis after recombinant tissue-type plasminogen activator (tPA) administration revealed that incomplete proteolysis of fibrin polymers markedly facilitated clot retraction. Similarly, inhibiting endogenous fibrinolysis with tranexamic acid reduced retraction of fibrin polymers in vivo. In vitro clot retraction experiments indicated that subthreshold doses of tPA facilitated clot retraction through a plasmin-dependent mechanism. These effects correlated with changes in the elastic modulus of fibrin clots. These findings define the endogenous fibrinolytic system as an important regulator of clot retraction, and show that promoting clot retraction is a novel and complementary means by which fibrinolytic enzymes can reduce thrombus size. © 2017 by The American Society of Hematology.

  6. In vivo human buccal permeability of nicotine

    DEFF Research Database (Denmark)

    Adrian, Charlotte L; Olin, Helle B D; Dalhoff, Kim

    2006-01-01

    The aim was to examine the in vivo buccal pH-dependent permeability of nicotine in humans and furthermore compare the in vivo permeability of nicotine to previous in vitro permeability data. The buccal permeability of nicotine was examined in a three-way cross-over study in eight healthy non......-smokers using a buccal perfusion cell. The disappearance of nicotine from perfusion solutions with pH 6.0, 7.4, and 8.1 was studied for 3h. The apparent permeability of nicotine (P(app)) was determined at each pH value. Parotid saliva was collected in an attempt to assess systemic levels of nicotine....... The disappearance rate of nicotine increased significantly as the pH increased, which resulted in P(app) values of 0.57+/-0.55 x 10(-4), 2.10+/-0.23 x 10(-4), and 3.96+/-0.54 x 10(-4)cms(-1) (mean+/-S.D.) at pH 6.0, 7.4, and 8.1, respectively. A linear relationship (R(2)=0.993) was obtained between the P...

  7. Experimental In Vivo Models of Candidiasis

    Directory of Open Access Journals (Sweden)

    Esther Segal

    2018-02-01

    Full Text Available Candidiasis is a multifaceted fungal disease including mucosal-cutaneous, visceral, and disseminated infections caused by yeast species of the genus Candida. Candida infections are among the most common human mycoses. Candida species are the third to fourth most common isolates from bloodstream infections in neutropenic or immunocompromised hospitalized patients. The mucosal-cutaneous forms—particularly vaginal infections—have a high prevalence. Vaginitis caused by Candida species is the second most common vaginal infection. Hence, candidiasis is a major subject for research, including experimental in vivo models to study pathogenesis, prevention, or therapy of the disease. The following review article will focus on various experimental in vivo models in different laboratory animals, such as mammals (mice, rats, rabbits, the fruit fly–Drosophila melanogaster, the larvae of the moth Galleria mellonella, or the free-living nematode Caenorhabditis elegans. The review will describe the induction of the different clinical forms of candidiasis in the various models and the validity of such models in mimicking the human clinical situations. The use of such models for the assessment of antifungal drugs, evaluation of potential vaccines to protect before candidiasis, exploration of Candida virulence factors, and comparison of pathogenicity of different Candida species will be included in the review. All of the above will be reported as based on published studies of numerous investigators as well as on the research of the author and his group.

  8. Does simvastatin stimulate bone formation in vivo?

    Directory of Open Access Journals (Sweden)

    Chorev Michael

    2003-04-01

    Full Text Available Abstract Background Statins, potent compounds that inhibit cholesterol synthesis in the liver have been reported to induce bone formation, both in tissue culture and in rats and mice. To re-examine potential anabolic effects of statins on bone formation, we compared the activity of simvastatin (SVS to the known anabolic effects of PTH in an established model of ovariectomized (OVX Swiss-Webster mice. Methods Mice were ovariectomized at 12 weeks of age (T0, remained untreated for 5 weeks to allow development of osteopenia (T5, followed by treatment for 8 weeks (T13. Whole, trabecular and cortical femoral bone was analyzed by micro-computed tomography (micro CT. Liquid chromatography/mass spectrometry (LC/MS was used to detect the presence of SVS and its active metabolite, simvastatin β-hydroxy acid (SVS-OH in the mouse serum. Results Trabecular BV/TV at T13 was 4.2 fold higher in animals treated with PTH (80 micro-g/kg/day compared to the OVX-vehicle treated group (p in vivo study. Conclusions While PTH demonstrated the expected anabolic effect on bone, SVS failed to stimulate bone formation, despite our verification by LC/MS of the active SVS-OH metabolite in mouse serum. While statins have clear effects on bone formation in vitro, the formulation of existing 'liver-targeted' statins requires further refinement for efficacy in vivo.

  9. Oxide nano crystals for in vivo imaging

    International Nuclear Information System (INIS)

    Heinrich, E.

    2005-01-01

    For small animal, fluorescence imaging is complementary with other techniques such as nuclear imaging (PET, SPECT). In vivo imaging studies imply the development of new luminescent probes, with a better sensitivity and a better biological targeting. These markers must filled biological and optical conditions. Our goal is to study new doped lanthanides oxide nano-crystals, their properties, their functionalization and their ability to target biological molecules. Characterizations of Y 2 O 3 :Eu and Y 2 SiO 5 :Eu nano-crystals (light diffusion, spectrometry, microscopy) allowed the determination of their size, their fluorescence properties but also their photo-bleaching. Means of stabilization of the nanoparticles were also studied in order to decrease their aggregation. Gd 2 O 3 :Eu nano-crystals were as well excited by X rays. Nano-crystals of Y 2 SiO 5 :Eu were functionalized, and organic ligands grafting evidenced by fluorescence and NMR. The functionalized nano-crystals could then recognized biological targets (streptavidin-biotin) and be incubated in the presence of HeLa cells. This report deals with the properties of these nano-crystals and their ability to meet the optical and biological conditions required for the application of in vivo imaging. (author)

  10. In vivo NMR spectroscopy of ripening avocado

    International Nuclear Information System (INIS)

    Bennett, A.B.; Smith, G.M.; Nichols, B.

    1987-01-01

    Ripening of avocado fruit is associated with a dramatic increase in respiration. Previous studies have indicated that the increase in respiration is brought about by activation of the glycolytic reaction catalyzing the conversion of fructose-6-phosphate to fructose 1,6-bisphosphate. The authors reinvestigated the proposed role of glycolytic regulation in the respiratory increase using in vivo 31 P nuclear magnetic resonance (NMR) spectroscopy using an external surface coil and analysis of phosphofructokinase (PFK), phosphofructophosphotransferase (PFP), and fructose 2,6-bisphosphate (fru 2,6-P 2 ) levels in ripening avocado fruit. In vivo 31 P NMR spectroscopy revealed large increases in ATP levels accompanying the increase in respiration. Both glycolytic enzymes, PFK and PFP, were present in avocado fruit, with the latter activity being highly stimulated by fru 2,6-P 2 . Fructose 2,6-bisphosphate levels increased approximately 90% at the onset of ripening, indicating that the respiratory increase in ripening avocado may be regulated by the activation of PFP brought about by an increase in fru 2,6-P 2

  11. Fluorescence based molecular in vivo imaging

    International Nuclear Information System (INIS)

    Ebert, Bernd

    2008-01-01

    Molecular imaging represents a modern research area that allows the in vivo study of molecular biological process kinetics using appropriate probes and visualization methods. This methodology may be defined- apart from the contrast media injection - as non-abrasive. In order to reach an in vivo molecular process imaging as accurate as possible the effects of the used probes on the biological should not be too large. The contrast media as important part of the molecular imaging can significantly contribute to the understanding of molecular processes and to the development of tailored diagnostics and therapy. Since more than 15 years PTB is developing optic imaging systems that may be used for fluorescence based visualization of tissue phantoms, small animal models and the localization of tumors and their predecessors, and for the early recognition of inflammatory processes in clinical trials. Cellular changes occur during many diseases, thus the molecular imaging might be of importance for the early diagnosis of chronic inflammatory diseases. Fluorescent dyes can be used as unspecific or also as specific contrast media, which allow enhanced detection sensitivity

  12. In vivo radiation sensitivity of glioblastoma multiforme

    International Nuclear Information System (INIS)

    Taghian, Alphonse; Freeman, Jill; Suit, Herman; DuBois, Willem; Budach, Wilfried; Baumann, Michael

    1995-01-01

    Purpose: Human glioblastoma (GBM) is one of the most resistant tumors to radiation. In previous reports, we have demonstrated a wide range of radiation sensitivity of GBM in vitro; that is, SF 2 values of 0.2 to 0.8. The great sensitivity of some of the cell lines is not in accord with the almost invariably fatal clinical outcome of patients with GBM. The sensitivity of cells in vitro pertains to cells cultured in optimal nutritional conditions. The TCD 50 (the radiation dose necessary to control 50% of the tumors locally) determined in lab animals is analogous to the use of radiation with curative intent in clinical radiation oncology. The aim of the present study was (a) to evaluate the sensitivity of GBM in vivo relative to that of other tumor types and (b) assess the relationship between the single dose TCD 50 of the xenografts and the sensitivity of the corresponding cell lines in vitro. Methods and Materials: The TCD 50 assay was used to study twelve human tumor lines. Four previously published values were added. A total of 10 GBM, 4 squamous cell carcinoma (SCC), 1 soft tissue sarcoma (STS), and 1 cancer colon (CC) are included in the analysis. For further suppression of the residual immune system, all the animals received 6 Gy whole-body irradiation 1 day before transplantation. Local tumor irradiations were given as a single dose, under conditions of clamp hypoxia using a Cs irradiator. Results: The TCD 50 values for the 10 GBM xenografts varied between 32.5 and 75.2 Gy, with an average of 47.2 ± 13.1 Gy. The TCD 50 values for the SCC were similar to those of the GBM and ranged from 40.7 and 54.4 Gy, with a mean of 46.8 ± 6.4. The difference between the average TCD 50 of GBM and SCC was not significant. The STS and CC xenografts had TCD 50 values of 46.0 and 49.2 Gy, respectively. No correlation was found between the TCD 50 in vivo and the SF 2 or D 0 in vitro. Conclusions: Our data on GBM xenografts showed a wide range of sensitivities to single dose

  13. In vivo radiation sensitivity of glioblastoma multiforme

    Energy Technology Data Exchange (ETDEWEB)

    Taghian, Alphonse; Freeman, Jill; Suit, Herman; DuBois, Willem; Budach, Wilfried; Baumann, Michael

    1995-04-30

    Purpose: Human glioblastoma (GBM) is one of the most resistant tumors to radiation. In previous reports, we have demonstrated a wide range of radiation sensitivity of GBM in vitro; that is, SF{sub 2} values of 0.2 to 0.8. The great sensitivity of some of the cell lines is not in accord with the almost invariably fatal clinical outcome of patients with GBM. The sensitivity of cells in vitro pertains to cells cultured in optimal nutritional conditions. The TCD{sub 50} (the radiation dose necessary to control 50% of the tumors locally) determined in lab animals is analogous to the use of radiation with curative intent in clinical radiation oncology. The aim of the present study was (a) to evaluate the sensitivity of GBM in vivo relative to that of other tumor types and (b) assess the relationship between the single dose TCD{sub 50} of the xenografts and the sensitivity of the corresponding cell lines in vitro. Methods and Materials: The TCD{sub 50} assay was used to study twelve human tumor lines. Four previously published values were added. A total of 10 GBM, 4 squamous cell carcinoma (SCC), 1 soft tissue sarcoma (STS), and 1 cancer colon (CC) are included in the analysis. For further suppression of the residual immune system, all the animals received 6 Gy whole-body irradiation 1 day before transplantation. Local tumor irradiations were given as a single dose, under conditions of clamp hypoxia using a Cs irradiator. Results: The TCD{sub 50} values for the 10 GBM xenografts varied between 32.5 and 75.2 Gy, with an average of 47.2 {+-} 13.1 Gy. The TCD{sub 50} values for the SCC were similar to those of the GBM and ranged from 40.7 and 54.4 Gy, with a mean of 46.8 {+-} 6.4. The difference between the average TCD{sub 50} of GBM and SCC was not significant. The STS and CC xenografts had TCD{sub 50} values of 46.0 and 49.2 Gy, respectively. No correlation was found between the TCD{sub 50} in vivo and the SF{sub 2} or D{sub 0} in vitro. Conclusions: Our data on GBM

  14. In vivo measurement of urethral dose profiles

    International Nuclear Information System (INIS)

    Toye, W.C.; Royal Melbourne Institute of Technology,; Duchesne, G.M.; Das, K.R.; Cee, A.; Mameghan, H.; Johnston, P.N.

    2001-01-01

    Full text: Quality assurance becomes a critical requirement when radiographs are routinely used in planning of treatments. In HDR prostate brachytherapy, the surrounding organs at risk of complications are the bladder and the rectum. However, of particular concern is the urethra that runs centrally through the prostate gland, as an unavoidably high dose can occur in the central region in order to achieve a minimum peripheral dose to a small target volume. Although high urethral doses have previously been related to increased urinary symptoms, some recent studies have not found such a correlation. The aim of this study was firstly, to identify dosimetric indicators of urethral morbidity following HDR prostate brachytherapy (4F x of 5.0 Gy), and secondly, to test the validity of calculated dose values. The in vivo measurements utilised a TLD (LiF:Mg,Ti) train formed by loading eight TLD rods alternating with 1,0 cm brass spacers into a fine plastic flexible tube. The length and diameter of plastic tubing was approximately 45cm and 0.15cm respectively, while the train length was 11.8 cm from the tip of the tube. The TLD train was placed into the central lumen of an 18 F three-way urethral catheter prior to its insertion. Significant urinary morbidity was defined prospectively as a score of a total of 3 or more points for severity from 5 symptoms categories. The five symptoms evaluated were hesitancy, frequency/nocturia, dysuria, haematuria and incontinence. The introduction of in vivo measurements to enhance the existing dosimetric analysis may be required to fully test the quantitative relationships (e.g. dose-volume ratios). Placement of TLDs within the urethra results in measurements whose accuracy is unaffected by internal organ motion as the hollow urethra must move with the prostate. The dose recorded by the TLDs is determined independently of the predictive algorithm used by the treatment planning system, and prostate location errors (e.g. due to image

  15. In vitro and in vivo toxicity assessment of nanoparticles

    Science.gov (United States)

    Kumar, Vinay; Sharma, Neha; Maitra, S. S.

    2017-11-01

    Nanotechnology has revolutionized gene therapy, diagnostics and environmental remediation. Their bulk production, uses and disposal have posed threat to the environment. With the appearance of these nanoparticles in the environment, their toxicity assessment is an immediate concern. This review is an attempt to summarize the major techniques used in cytotoxity determination. The review also presents a detailed and elaborative discussion on the toxicity imposed by different types of nanoparticles including carbon nanotubes, gold nanoparticles, silver nanoparticles, quantum dots, fullerenes, aluminium nanoparticles, zinc nanoparticles, iron nanoparticles, titanium nanoparticles and silica nanoparticles. It discusses the in vitro and in vivo toxological effects of nanoparticles on bacteria, microalgae, zebrafish, crustacean, fish, rat, mouse, pig, guinea pig, human cell lines and human. It also discusses toxological effects on organs such as liver, kidney, spleen, sperm, neural tissues, liver lysosomes, spleen macrophages, glioblastoma cells, hematoma cells and various mammalian cell lines. It provides information about the effects of nanoparticles on the gene-expression, growth and reproduction of the organisms.

  16. In vivo somatic mutation systems in the mouse

    International Nuclear Information System (INIS)

    Russell, L.B.

    1979-01-01

    In an effort to meet the need for a fast and cheap in vivo prescreen for inherited mammalian point mutations, a somatic forward-mutation method, originally developed in an x-ray experiment, has more recently been tested in work with chemical mutagens. The method makes use of coat-color mutations because the gene product is usually locally expressed, mosaics can be detected with minimal effort, and opportunities for making comparison with induction of germinal point mutations are greatest.--Following treatment of embryos that are heterozygous at specific coat-color loci, various induced genetic changes can result in expression of the recessive (RS) in clones derived from mutant melanocyte precursor cells. However, other events, such as decrease in the number of precursor cells, or disturbed differentiation, can also result in spots, which with careful classification can usually be distinguished from RS's on the basis of their location and color. When this is done, the relative RS frequencies for a series of compounds at least roughly parallel the relative spermatogonial mutation rates. The fact that easily measurable (though low) RS rates are obtained with compounds that have yielded negative results in spermatogonial tests is not surprising in view of the fact that RS's can be caused by several mechanisms besides point mutation.--In spite of the parallelism observed in one laboratory, the usefulness of the in vivo somatic mutation method as a prescreen could come to be doubted because of major discrepancies between results of similar experiments at different laboratories. However, It appears probable that at least some of these discrepancies are due to failure to discriminate between spots that probably resulted from melanocyte insufficiency and spots that resulted from expression of the recessive.--Reverse somatic mutation systems can potentially avoid some of the pitfalls of forward mutation systems. Such system are still in developmental stages

  17. In vivo dosimetry in radiation therapy in Sweden; In vivo-dosimetri inom straalbehandling i Sverige

    Energy Technology Data Exchange (ETDEWEB)

    Eriksson, Jacob; Blomquist, Michael (Norrlands universitetssjukhus, Umeaa (Sweden))

    2010-07-15

    A prerequisite for achieving high radiation safety for patients receiving external beam radiation therapy is that the hospitals have a quality assurance program. The program should include include monitoring of the radiation dose given to the patient. Control measurements are performed both at the system level and at the individual level. Control measurement is normally performed using in vivo dosimetry, e.g. a method to measure the radiation dose at the individual level during the actual radiation treatment time. In vivo dosimetry has proven to be an important tool to detect and prevent serious errors in patient treatment. The purpose of this research project was to identify the extent to which vivo dosimetry is used and the methods available for this at Swedish radiation therapy clinics. The authority also wanted to get an overall picture of how hospitals manage results of in vivo dosimetry, and how clinics control radiation dose when using modern treatment techniques. The report reflects the situation in Swedish radiotherapy clinics 2007. The report shows that all hospitals use some form of in vivo dosimetry. The instruments used are mainly diodes and termoluminiscence dosimeters

  18. ST37 Klebsiella pneumoniae: development of carbapenem resistance in vivo during antimicrobial therapy in neonates.

    Science.gov (United States)

    Li, Pengling; Wang, Min; Li, Xianping; Hu, Feihu; Yang, Min; Xie, Yixin; Cao, Wei; Xia, Xiaomeng; Zheng, Rong; Tian, Jingjing; Zhang, Kan; Chen, Fang; Tang, Aiguo

    2017-08-01

    To investigate the mechanism leading to in vivo carbapenem resistance development in Klebsiella pneumoniae. Carbapenemase was detected using the modified carbapenem inactivation method. β-lactamases resistant genes were identified by PCR and sequencing. Clonal relatedness was evaluated by random amplified polymorphic DNA and multiple locus sequence typing. The relationship between sequence typing and resistant genes was analyzed by using the chi-squared test. All ST37 carbapenem-resistant isolates were bla OXA-1 positive and all ST37 carbapenem-sensitive isolates were bla OXA-1 negative at Stage I. A significant relationship between carbapenem resistance and bla OXA-1 was observed. The bla OXA-1 -positive rate was significantly higher in ST37 K. pneumoniae than others. This is the first study about the development of carbapenem resistance in vivo potentially mediated by bla OXA-1 in ST37 K. pneumoniae among neonates.

  19. Radon and its daughters in vivo

    International Nuclear Information System (INIS)

    Rundo, J.

    1983-01-01

    Some aspects of the behavior of radon and its short-lived daughters in vivo are described and a relationship between the radon exhalation rate and time after a meal is demonstrated. A major but short-lived postprandial increase in the exhalation rate of radon produced from skeletally-deposited radium was observed and a similar effect in exhalation rate of environmental radon by persons containing no radium was noted. Persons living in houses with elevated concentrations of radon may contain sufficient activity for its detection by external gamma-ray counting. Some of the activity observed is due to inhaled daughter-products in the chest, and some to daughter-products associated with and produced by the decay of radon throughout the body. 3 references, 8 figures. (MF)

  20. In Vivo Cytogenetic Studies on Aspartame

    Directory of Open Access Journals (Sweden)

    Entissar S. AlSuhaibani

    2010-01-01

    Full Text Available Aspartame (a-Laspartyl-L-phenylalanine 1-methylester is a dipeptide low-calorie artificial sweetener that is widely used as a nonnutritive sweetener in foods and drinks. The safety of aspartame and its metabolic breakdown products (phenylalanine, aspartic acid and methanol was investigated in vivo using chromosomal aberration (CA test and sister chromatid exchange (SCE test in the bone marrow cells of mice. Swiss Albino male mice were exposed to aspartame (3.5, 35, 350 mg/kg body weight. Bone marrow cells isolated from femora were analyzed for chromosome aberrations and sister chromatid exchanges. Treatment with aspartame induced dose dependently chromosome aberrations at all concentrations while it did not induce sister chromatid exchanges. On the other hand, aspartame did not decrease the mitotic index (MI. However, statistical analysis of the results show that aspartame is not significantly genotoxic at low concentration.

  1. Chemical labeling of gluatmate decarboxylase in vivo

    International Nuclear Information System (INIS)

    Rando, R.R.

    1981-01-01

    Mouse brain glutamate decarboxylase(s) was specifically titrated in vivo and in crude brain homogenates by a combination of gabaculine and [alpha-3H]acetylenic gamma-aminobutyric acid. This specific titration is based on the differential spectra of action of these two mechanism-based enzyme inactivators. The specificity of the titration in vitro was demonstrated by showing that the time course of radioactivity incorporation exactly paralleled the time course for glutamate of decarboxylase inactivation. This means that there is approximately 0.66 nmol of glutamate decarboxylase/0.5 g of mouse brain, assuming the stoichiometry of inactivator bound to enzyme is one. This value is similar to the one obtained from a calculation based on the enzyme purification data

  2. Quantifying drug-protein binding in vivo

    International Nuclear Information System (INIS)

    Buchholz, B; Bench, G; Keating III, G; Palmblad, M; Vogel, J; Grant, P G; Hillegonds, D

    2004-01-01

    Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS

  3. NMR studies of cerebral metabolism in vivo

    International Nuclear Information System (INIS)

    Prichard, J.W.

    1986-01-01

    The nature and extent of the potential synergism between PET and NMR methods is not yet well appreciated in the biomedical community. The long-range interest of medical neurobiology will be well served by efforts of PET and NMR scientists to follow each others' work so that opportunities for productive interchange can be efficiently exploited. Appreciation of the synergism by the rest of the biomedical community will follow naturally. PET is said by the people doing it to be still in its infancy, for they are more concerned with advancing their discipline than with admiring its already impressive achievements. On the scale of the same developmental metaphor, many NMR methods for studying the living human brain are still in utero. The best way to provide the reader a sense of the current status and future course of NMR research in medical neurobiology is by discussion of published in vivo studies. Such a discussion, adapted from another article is what follows

  4. Techniques of in vivo neutron activation analysis

    International Nuclear Information System (INIS)

    Chettle, D.R.; Fremlin, J.H.

    1984-01-01

    This review is dealt with under the following headings, intended to reflect the different factors affecting the measurement sensitivity, starting with the choice of neutron source and proceeding, through the reaction characteristics, to the detection system, the questions of dosimetry and ethical constraints being also discussed: 1) neutron sources, slowing down and interaction processes, energy spectrum and flux uniformity, timing 2) neutron reactions used for in vivo analyses 3) detectors, choice, geometrical considerations and detector shielding 4) data collection and processing 5) interpretation, major elements, absolute or sequential measurements, relationship to other parameters 6) dosimetry, framework for dose levels, biological effects of neutron interactions, neutron doses in practice 7) implications for measurement of calcium, nitrogen and cadmium. (U.K.)

  5. Radon and its daughters in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Rundo, J

    1984-05-01

    Prolonged exposure to radon should build up a reservoir of radon in body fat and fluids. If the subject moved to an environment with a lower radon concentration from an environment with a higher level of radon, the result would be an exhalation of radon, and the initial exhalation rate of radon should depend of the radon concentration inhaled. This paper describes the behavior of radon and its daughters in vivo and a relationship between the radon exhalation rate and the time after a meal. A major but short-lived postprandial increase in the exhalation rate of radon was observed. The author reports a similar effect in the exhalation rate of radon by persons containing no radium. It should be noted that the possibility exists that a large amount of radon daughters in the chest may interfere in the investigation of possible internal contamination with plutonium or other actinides by external counting. 8 figures.

  6. Radon and its daughters in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Rundo, J

    1984-05-01

    Prolonged exposure to radon should build up a reservoir of radon in body fat and fluids. Movement of the subject to an environment with a lower radon concentration from an environment with a higher level of radon would result in an exhalation of radon, and the initial exhalation rate of radon should depend on the radon concentration inhaled. This paper describes the behavior of radon and its daughters in vivo and a relationship between the radon exhalation rate and the time after a meal. A major but short-lived postprandial increase in the exhalation rate of radon was observed. We report a similar effect in the exhalation rate of radon by persons containing no radium. It should be noted that the possibility exists that a large amount of radon daughters in the chest may interfere in the investigation of possible internal contamination with plutonium or other actinides by external counting. (author).

  7. Implantable optoelectronic probes for in vivo optogenetics

    Science.gov (United States)

    Iseri, Ege; Kuzum, Duygu

    2017-06-01

    More than a decade has passed since optics and genetics came together and lead to the emerging technologies of optogenetics. The advent of light-sensitive opsins made it possible to optically trigger the neurons into activation or inhibition by using visible light. The importance of spatiotemporally isolating a segment of a neural network and controlling nervous signaling in a precise manner has driven neuroscience researchers and engineers to invest great efforts in designing high precision in vivo implantable devices. These efforts have focused on delivery of sufficient power to deep brain regions, while monitoring neural activity with high resolution and fidelity. In this review, we report the progress made in the field of hybrid optoelectronic neural interfaces that combine optical stimulation with electrophysiological recordings. Different approaches that incorporate optical or electrical components on implantable devices are discussed in detail. Advantages of various different designs as well as practical and fundamental limitations are summarized to illuminate the future of neurotechnology development.

  8. In vivo nuclear imaging of apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tae Sup; Cheon, Gi Jeong [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2004-04-01

    Apoptosis plays a role in the pathophysiology of many kinds of diseases and in the response of treatment. Compared to the necrosis, the apoptosis a genetically controlled and energy-dependent process which removes the unwanted cells from the body; programmed cell death or cell suicide. During the apoptosis, phosphatidylserine is expressed in the cytoplasmic outer membrane in the early phase. Annexin V, an endogenous human protein (MW=35 kD), has an affinity of about 10{sup -9} M for the phosphatidylserine exposed on the outer membrane of apoptotic cells. Annexin V can be radiolabeled with {sup 99}mTc by HYNIC or EC chelators, which can be used as an radiotracer for the in vivo imaging of apoptosis. In this article, we reviewed the apoptosis, radiolabeling of annexin V, and the experimental and clinical data using annexin V imaging.

  9. Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system.

    Science.gov (United States)

    Heppert, Jennifer K; Dickinson, Daniel J; Pani, Ariel M; Higgins, Christopher D; Steward, Annette; Ahringer, Julie; Kuhn, Jeffrey R; Goldstein, Bob

    2016-11-07

    Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments. © 2016 Heppert et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  10. In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters.

    OpenAIRE

    Buckley, SM; Delhove, JM; Perocheau, DP; Karda, R; Rahim, AA; Howe, SJ; Ward, NJ; Birrell, MA; Belvisi, MG; Arbuthnot, P; Johnson, MR; Waddington, SN; McKay, TR

    2015-01-01

    The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruse...

  11. Targeted detection of murine colonic dysplasia in vivo with flexible multispectral scanning fiber endoscopy

    Science.gov (United States)

    Joshi, Bishnu P.; Miller, Sharon J.; Lee, Cameron; Gustad, Adam; Seibel, Eric J.; Wang, Thomas D.

    2012-02-01

    We demonstrate a multi-spectral scanning fiber endoscope (SFE) that collects fluorescence images in vivo from three target peptides that bind specifically to murine colonic adenomas. This ultrathin endoscope was demonstrated in a genetically engineered mouse model of spontaneous colorectal adenomas based on somatic Apc (adenomatous polyposis coli) gene inactivation. The SFE delivers excitation at 440, 532, 635 nm with human patients by simultaneously visualizing multiple over expressed molecular targets unique to dysplasia.

  12. In vivo transcriptional profiling of Listeria monocytogenes and mutagenesis identify new virulence factors involved in infection.

    Directory of Open Access Journals (Sweden)

    Ana Camejo

    2009-05-01

    Full Text Available Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host-pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. In the spleen of infected mice, approximately 20% of the Listeria genome is differentially expressed, essentially through gene activation, as compared to exponential growth in rich broth medium. Data presented here show that, during infection, Listeria is in an active multiplication phase, as revealed by the high expression of genes involved in replication, cell division and multiplication. In vivo bacterial growth requires increased expression of genes involved in adaptation of the bacterial metabolism and stress responses, in particular to oxidative stress. Listeria interaction with its host induces cell wall metabolism and surface expression of virulence factors. During infection, L. monocytogenes also activates subversion mechanisms of host defenses, including resistance to cationic peptides, peptidoglycan modifications and release of muramyl peptides. We show that the in vivo differential expression of the Listeria genome is coordinated by a complex regulatory network, with a central role for the PrfA-SigB interplay. In particular, L. monocytogenes up regulates in vivo the two major virulence regulators, PrfA and VirR, and their downstream effectors. Mutagenesis of in vivo induced genes allowed the identification of novel L. monocytogenes virulence factors, including an LPXTG surface protein, suggesting a role for S-layer glycoproteins and for cadmium efflux system in Listeria virulence.

  13. In vivo nanotoxicity assays in plant models.

    Science.gov (United States)

    Kumari, Mamta; Ernest, Vinita; Mukherjee, Amitava; Chandrasekaran, Natarajan

    2012-01-01

    Increasing application of silver nanoparticles (SNPs) and zinc oxide nanoparticles (nZnO) in consumer products like textiles, cosmetics, washing machines and other household products increases their chance to reach the environment. Intensive research is required to assess the nanoparticles' toxicity to the environmental system. The toxicological effect of nanoparticles has been studied at the miniscule scale and requires intensive research to be conducted to assess its unknown effects. Plants are the primary target species which need to be included to develop a comprehensive toxicity profile for nanoparticles. So far, the mechanisms of toxicity of nanoparticles to the plant system remains largely unknown and little information on the potential uptake of nanoparticles by plants and their subsequent fate within the food chain is available. The phytoxicological behaviour of silver and zinc oxide nanoparticles on Allium cepa and seeds of Zea mays (maize), Cucumis sativus (cucumber) and Lycopersicum esculentum (tomato) was done. The in vitro studies on A. cepa have been done to check the cytotoxicological effects including mitotic index, chromosomal aberrations, vagrant chromosomes, sticky chromosomes, disturbed metaphase, breaks and formation of micronucleus. In vitro and in vivo studies on seed systems exposed to different concentration of nanoparticles dispersion to check phytotoxicity end point as root length, germination effect, adsorption and accumulation of nanoparticles (uptake studies) into the plant systems. In vivo studies in a seed system was done using phytagel medium. Biochemical studies were done to check effect on protein, DNA and thiobarbituric acid reactive species concentration. FT-IR studies were done to analyze the functional and conformational changes in the treated and untreated samples. The toxicological effects of nanoparticles had to be studied at the miniscule scale to address existing environment problems or prevent future problems. The

  14. In vivo Raman spectroscopy of cervix cancers

    Science.gov (United States)

    Rubina, S.; Sathe, Priyanka; Dora, Tapas Kumar; Chopra, Supriya; Maheshwari, Amita; Krishna, C. Murali

    2014-03-01

    Cervix-cancer is the third most common female cancer worldwide. It is the leading cancer among Indian females with more than million new diagnosed cases and 50% mortality, annually. The high mortality rates can be attributed to late diagnosis. Efficacy of Raman spectroscopy in classification of normal and pathological conditions in cervix cancers on diverse populations has already been demonstrated. Our earlier ex vivo studies have shown the feasibility of classifying normal and cancer cervix tissues as well as responders/non-responders to Concurrent chemoradiotherapy (CCRT). The present study was carried out to explore feasibility of in vivo Raman spectroscopic methods in classifying normal and cancerous conditions in Indian population. A total of 182 normal and 132 tumor in vivo Raman spectra, from 63 subjects, were recorded using a fiberoptic probe coupled HE-785 spectrometer, under clinical supervision. Spectra were acquired for 5 s and averaged over 3 times at 80 mW laser power. Spectra of normal conditions suggest strong collagenous features and abundance of non-collagenous proteins and DNA in case of tumors. Preprocessed spectra were subjected to Principal Component-Linear Discrimination Analysis (PCLDA) followed by leave-one-out-cross-validation. Classification efficiency of ~96.7% and 100% for normal and cancerous conditions respectively, were observed. Findings of the study corroborates earlier studies and suggest applicability of Raman spectroscopic methods in combination with appropriate multivariate tool for objective, noninvasive and rapid diagnosis of cervical cancers in Indian population. In view of encouraging results, extensive validation studies will be undertaken to confirm the findings.

  15. Fetal microglial in vitro phenotype depends on prior in vivo inflammation

    Directory of Open Access Journals (Sweden)

    Mingju eCao

    2015-08-01

    Full Text Available Objective. Neuroinflammation in utero may result in life-long neurological disabilities. The molecular mechanisms whereby microglia contribute to this response remain incompletely understood. Methods. Lipopolysaccharide (LPS or saline were administered intravenously to non-anesthetized chronically instrumented near-term fetal sheep to model fetal inflammation in vivo. Microglia were then isolated from in vivo LPS and saline (naïve exposed animals. To mimic the second hit of neuroinflammation, these microglia were then re-exposed to LPS in vitro. Cytokine responses were measured in vivo and subsequently in vitro in the primary microglia cultures derived from these animals. We sequenced the whole transcriptome of naïve and second hit microglia and profiled their genetic expression to define molecular pathways disrupted during neuroinflammation.Results. In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure. Even though not histologically apparent, microglia acquired a pro-inflammatory phenotype in vivo that was sustained and amplified in vitro upon second hit LPS exposure as measured by IL-1β response in vitro and RNAseq analyses. While NFKB and Jak-Stat inflammatory pathways were up regulated in naïve microglia, heme oxygenase 1 (HMOX1 and Fructose-1,6-bisphosphatase (FBP genes were uniquely differentially expressed in the second hit microglia. Microglial calreticulin/LRP genes implicated in microglia-neuronal communication relevant for the neuronal development were up regulated in second hit microglia.Discussion. We identified a unique HMOX1down and FBPup phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways as a memory of prior inflammatory insult. These findings suggest new therapeutic targets for early postnatal intervention to prevent brain injury.

  16. In vivo bioluminescence imaging of cell differentiation in biomaterials: a platform for scaffold development.

    Science.gov (United States)

    Bagó, Juli R; Aguilar, Elisabeth; Alieva, Maria; Soler-Botija, Carolina; Vila, Olaia F; Claros, Silvia; Andrades, José A; Becerra, José; Rubio, Nuria; Blanco, Jerónimo

    2013-03-01

    In vivo testing is a mandatory last step in scaffold development. Agile longitudinal noninvasive real-time monitoring of stem cell behavior in biomaterials implanted in live animals should facilitate the development of scaffolds for tissue engineering. We report on a noninvasive bioluminescence imaging (BLI) procedure for simultaneous monitoring of changes in the expression of multiple genes to evaluate scaffold performance in vivo. Adipose tissue-derived stromal mensenchymal cells were dually labeled with Renilla red fluorescent protein and firefly green fluorescent protein chimeric reporters regulated by cytomegalovirus and tissue-specific promoters, respectively. Labeled cells were induced to differentiate in vitro and in vivo, by seeding in demineralized bone matrices (DBMs) and monitored by BLI. Imaging results were validated by RT-polymerase chain reaction and histological procedures. The proposed approach improves molecular imaging and measurement of changes in gene expression of cells implanted in live animals. This procedure, applicable to the simultaneous analysis of multiple genes from cells seeded in DBMs, should facilitate engineering of scaffolds for tissue repair.

  17. Transcriptional mapping of rabies virus in vivo

    International Nuclear Information System (INIS)

    Flamand, A.; Delagneau, J.F.

    1978-01-01

    Synthesis of the proteins of rabies virus was studied in hamster cell infected with uv-irradiated virus. The uv target size of genes L, N, M 1 , and M 2 was measured during primary transcription. Except for N, the target size of the remaining genes was considerably larger than that of their physical sizes. The data fit the hypothesis that four genes occupy a single transcriptional unit and that transcription of rabies virus proceeds in the order N, M 1 , M 2 , and L

  18. Expression of RANKL/OPG during bone remodeling in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, H., E-mail: tnk@ymghp.jp [Department of Orthopedic Surgery, Yamaguchi Grand Medical Center, 77 Ohsaki, Hofu, Yamaguchi 747-8511 (Japan); Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224 (United States); Mine, T. [Department of Orthopedic Surgery, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505 (Japan); Ogasa, H. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224 (United States); Department of Orthopedic Surgery, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505 (Japan); Taguchi, T. [Department of Orthopedic Surgery, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505 (Japan); Liang, C.T. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224 (United States); National Health Research Institutes, Taipei 115, Taiwan (China)

    2011-08-12

    Highlights: {yields} This is the first study to determine the relationship between osteogenic differentiation and RANKL/OPG expression during bone remodeling in vivo. {yields} The OPG expression peak occurred during the bone formation phase, whereas the marked elevation of RANKL expression was observed during the bone resorption phase. {yields} Histological analysis showed that RANKL/OPG immunoreactivity was predominantly associated with bone marrow cells in the marrow cavity. {yields} The present study confirmed that RANKL/OPG are key factors linking bone formation to resorption during the bone remodeling process. -- Abstract: The interaction between receptor activator of nuclear factor {kappa}B ligand (RANKL) and osteoprotegerin (OPG) plays a dominant role in osteoclastogenesis. As both proteins are produced by osteoblast lineage cells, they are considered to represent a key link between bone formation and resorption. In this study, we investigated the expression of RANKL and OPG during bone remodeling in vivo to determine the relationship between osteoclastogenic stimulation and osteoblastic differentiation. Total RNA was prepared from rat femurs after marrow ablation on days 0, 3, 6, and 9. The temporal activation patterns of osteoblast-related genes (procollagen {alpha}1 (I), alkaline phosphatase, osteopontin, and osteocalcin) were examined by Northern blot analysis. An appreciable increase in the expression of these osteoblast markers was observed on day 3. The peak increase in gene expression was observed on day 6 followed by a slight reduction by day 9. Real-time PCR analysis showed that the OPG mRNA expression was markedly upregulated on day 6 and slightly decreased on day 9. In contrast, RANKL mRNA expression was increased by more than 20-fold on day 9. The RANKL/OPG ratio, an index of osteoclastogenic stimulation, peaked on day 9. Histological analysis showed that RANKL and OPG immunoreactivity were predominantly associated with bone marrow cells. The

  19. Expression of RANKL/OPG during bone remodeling in vivo

    International Nuclear Information System (INIS)

    Tanaka, H.; Mine, T.; Ogasa, H.; Taguchi, T.; Liang, C.T.

    2011-01-01

    Highlights: → This is the first study to determine the relationship between osteogenic differentiation and RANKL/OPG expression during bone remodeling in vivo. → The OPG expression peak occurred during the bone formation phase, whereas the marked elevation of RANKL expression was observed during the bone resorption phase. → Histological analysis showed that RANKL/OPG immunoreactivity was predominantly associated with bone marrow cells in the marrow cavity. → The present study confirmed that RANKL/OPG are key factors linking bone formation to resorption during the bone remodeling process. -- Abstract: The interaction between receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) plays a dominant role in osteoclastogenesis. As both proteins are produced by osteoblast lineage cells, they are considered to represent a key link between bone formation and resorption. In this study, we investigated the expression of RANKL and OPG during bone remodeling in vivo to determine the relationship between osteoclastogenic stimulation and osteoblastic differentiation. Total RNA was prepared from rat femurs after marrow ablation on days 0, 3, 6, and 9. The temporal activation patterns of osteoblast-related genes (procollagen α1 (I), alkaline phosphatase, osteopontin, and osteocalcin) were examined by Northern blot analysis. An appreciable increase in the expression of these osteoblast markers was observed on day 3. The peak increase in gene expression was observed on day 6 followed by a slight reduction by day 9. Real-time PCR analysis showed that the OPG mRNA expression was markedly upregulated on day 6 and slightly decreased on day 9. In contrast, RANKL mRNA expression was increased by more than 20-fold on day 9. The RANKL/OPG ratio, an index of osteoclastogenic stimulation, peaked on day 9. Histological analysis showed that RANKL and OPG immunoreactivity were predominantly associated with bone marrow cells. The expression of bone formation

  20. Targeted cancer gene therapy : the flexibility of adenoviral gene therapy vectors

    NARCIS (Netherlands)

    Rots, MG; Curiel, DT; Gerritsen, WR; Haisma, HJ

    2003-01-01

    Recombinant adenoviral vectors are promising reagents for therapeutic interventions in humans, including gene therapy for biologically complex diseases like cancer and cardiovascular diseases. In this regard, the major advantage of adenoviral vectors is their superior in vivo gene transfer

  1. In Vivo Monitoring Program Manual, PNL-MA-574

    Energy Technology Data Exchange (ETDEWEB)

    Lynch, Timothy P.

    2010-07-01

    An overview of the administration for the In Vivo Monitoring Program (IVMP) for Hanford. This includes organizational structure and program responsibilities; coordination of in vivo measurements; scheduling measurements; performing measurements; reporting results; and quality assurance. Overall responsibility for the management of the IVMP rests with the Program Manager (PM). The PM is responsible for providing the required in vivo counting services for Hanford Site contractor employees in accordance with Department of Energy (DOE) requirements and the specific statements of work.

  2. In vivo analysis of the Notch receptor S1 cleavage.

    Directory of Open Access Journals (Sweden)

    Robert J Lake

    2009-08-01

    Full Text Available A ligand-independent cleavage (S1 in the extracellular domain of the mammalian Notch receptor results in what is considered to be the canonical heterodimeric form of Notch on the cell surface. The in vivo consequences and significance of this cleavage on Drosophila Notch signaling remain unclear and contradictory. We determined the cleavage site in Drosophila and examined its in vivo function by a transgenic analysis of receptors that cannot be cleaved. Our results demonstrate a correlation between loss of cleavage and loss of in vivo function of the Notch receptor, supporting the notion that S1 cleavage is an in vivo mechanism of Notch signal control.

  3. Passive in vivo elastography from skeletal muscle noise

    International Nuclear Information System (INIS)

    Sabra, Karim G.; Conti, Stephane; Roux, Philippe; Kuperman, W. A.

    2007-01-01

    Measuring the in vivo elastic properties of muscles (e.g., stiffness) provides a means for diagnosing and monitoring muscular activity. The authors demonstrated a passive in vivo elastography technique without an active external radiation source. This technique instead uses cross correlations of contracting skeletal muscle noise recorded with skin-mounted sensors. Each passive sensor becomes a virtual in vivo shear wave source. The results point to a low-cost, noninvasive technique for monitoring biomechanical in vivo muscle properties. The efficacy of the passive elastography technique originates from the high density of cross paths between all sensor pairs, potentially achieving the same sensitivity obtained from active elastography methods

  4. Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo

    DEFF Research Database (Denmark)

    Nudelman, Aaron Samuel; DiRocco, Derek P; Lambert, Talley J

    2010-01-01

    Activity-dependent changes in gene-expression are believed to underlie the molecular representation of memory. In this study, we report that in vivo activation of neurons rapidly induces the CREB-regulated microRNA miR-132. To determine if production of miR-132 is regulated by neuronal activity its......, olfactory bulb, and striatum by contextual fear conditioning, odor-exposure, and cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics of pri-miR-132 were monitored and found to parallel those of immediate early genes, peaking at 45 min and returning to basal levels within 2 h...

  5. Nanomedicine for Inner Ear Diseases: A Review of Recent In Vivo Studies

    Directory of Open Access Journals (Sweden)

    Dong-Kee Kim

    2017-01-01

    Full Text Available Nanoparticles are promising therapeutic options for inner ear disease. In this report, we review in vivo animal studies in the otologic field using nanoparticles over the past 5 years. Many studies have used nanoparticles to deliver drugs, genes, and growth factors, and functional and morphological changes have been observed. The constituents of nanoparticles are also diversifying into various biocompatible materials, including poly(lactic-co-glycolic acid (PLGA. The safe and effective delivery of drugs or genes in the inner ear will be a breakthrough for the treatment of inner ear diseases, including age-related hearing loss.

  6. Identifying A Molecular Phenotype for Bone Marrow Stromal Cells With In Vivo Bone Forming Capacity

    DEFF Research Database (Denmark)

    Larsen, Kenneth H; Frederiksen, Casper M; Burns, Jorge S

    2009-01-01

    (17% versus 5%) and a larger percentage of genes with predicted SP3 transcription factor binding sites in their promoter region (21% versus 8%). On the other hand, hBMSC-TERT(-Bone) cells expressed a larger number of immune-response related genes (26% versus 8%). In order to test for the predictive...... value of these markers, we studied the correlation between their expression levels in 6 different hBMSC-derived clones and the ability to form bone in vivo. We found a significant correlation for, decorin, lysyl oxidase-like 4, natriuretic peptide receptor C, and tetranectin. No significant positive...

  7. Antiandrogenic effects in short-term in vivo studies of the fungicide fenarimol

    DEFF Research Database (Denmark)

    Vinggaard, Anne; Jacobsen, H.; Metzdorff, Stine Broeng

    2005-01-01

    of ventral prostate, seminal vesicles. musc. levator anitbulbocavernosus, and bulbourethral glands. Qualitatively similar, but weaker, effects were also evident in intact fenarimol-exposed young adult males. except that prostates were not significantly affected. Changes in androgen-regulated gene expression...... that fenarimol acts as an antiandrogen in vivo having effects qualitatively comparable to those of flutamide on organ level, whereas differential effects on gene expression were observed. In an additional Hershberger test, the effects of fenarimol were compared to those of estradiol benzoate, prochloraz...

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

    Science.gov (United States)

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

    2014-11-01

    Development of an oral in vivo predictive dissolution medium for acid drugs with a pKa in the physiological range (e.g., Biopharmaceutics Classification System Class IIa) requires transport analysis of the complex in vivo CO2 /bicarbonate buffering system. In this report, we analyze this buffer system using hydrodynamically defined rotating disk dissolution. Transport analysis of drug flux was predicted using the film model approach of Mooney et al based on equilibrium assumptions as well as accounting for the slow hydration reaction, CO2 + H2 O → H2 CO3 . The accuracy of the models was compared with experimentally determined results using the rotating disk dissolution of ibuprofen, indomethacin, and ketoprofen. The equilibrium and slow hydration reaction rate models predict significantly different dissolution rates. The experimental results are more accurately predicted by accounting for the slow hydration reaction under a variety of pH and hydrodynamic conditions. Although the complex bicarbonate buffering system requires further consideration given its dynamic nature in vivo, a simplifying irreversible reaction (IRR) transport analysis accurately predicts in vitro rotating disk dissolution rates of several carboxylic acid drugs. This IRR transport model provides further insight into bicarbonate buffer and can be useful in developing more physiologically relevant buffer systems for dissolution testing. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-02-01

    In vivo bioluminescence imaging (BLI) is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating tumor growth. In this study, the kinetic of tumor growth has been assessed in C26 colon carcinoma bearing BALB/c mouse model. The ability of BLI to noninvasively quantitate the growth of subcutaneous tumors transplanted with C26 cells genetically engineered to stably express firefly luciferase and herpes simplex virus type-1 thymidine kinase (C26/tk-luc). A good correlation (R {sup 2}=0.998) of photon emission to the cell number was found in vitro. Tumor burden and tumor volume were monitored in vivo over time by quantitation of photon emission using Xenogen IVIS 50 and standard external caliper measurement, respectively. At various time intervals, tumor-bearing mice were imaged to determine the correlation of in vivo BLI to tumor volume. However, a correlation of BLI to tumor volume was observed when tumor volume was smaller than 1000 mm{sup 3} (R {sup 2}=0.907). {gamma} Scintigraphy combined with [{sup 131}I]FIAU was another imaging modality used for verifying the previous results. In conclusion, this study showed that bioluminescence imaging is a powerful and quantitative tool for the direct assay to monitor tumor growth in vivo. The dual reporter genes transfected tumor-bearing animal model can be applied in the evaluation of the efficacy of new developed anti-cancer drugs.

  10. DNA moves sequentially towards the nuclear matrix during DNA replication in vivo

    Directory of Open Access Journals (Sweden)

    Aranda-Anzaldo Armando

    2011-01-01

    Full Text Available Abstract Background In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM. There is varied evidence indicating that DNA replication occurs in replication factories organized upon the NM and that DNA loops may correspond to the actual replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but they synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration in vivo. We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops. Results In the present work we tracked down the movement relative to the NM of DNA sequences located at different points within such five structural DNA loops during the S phase and after the return to cellular quiescence during liver regeneration. Our results indicate that looped DNA moves sequentially towards the NM during replication and then returns to its original position in newly quiescent cells, once the liver regeneration has been achieved. Conclusions Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated. These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.

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

    International Nuclear Information System (INIS)

    Chen, C.-C.; Hwang, Jeng-Jong; Ting, G.; Tseng, Y.-L.; Wang, S.-J.; Whang-Peng, J.

    2007-01-01

    In vivo bioluminescence imaging (BLI) is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating tumor growth. In this study, the kinetic of tumor growth has been assessed in C26 colon carcinoma bearing BALB/c mouse model. The ability of BLI to noninvasively quantitate the growth of subcutaneous tumors transplanted with C26 cells genetically engineered to stably express firefly luciferase and herpes simplex virus type-1 thymidine kinase (C26/tk-luc). A good correlation (R 2 =0.998) of photon emission to the cell number was found in vitro. Tumor burden and tumor volume were monitored in vivo over time by quantitation of photon emission using Xenogen IVIS 50 and standard external caliper measurement, respectively. At various time intervals, tumor-bearing mice were imaged to determine the correlation of in vivo BLI to tumor volume. However, a correlation of BLI to tumor volume was observed when tumor volume was smaller than 1000 mm 3 (R 2 =0.907). γ Scintigraphy combined with [ 131 I]FIAU was another imaging modality used for verifying the previous results. In conclusion, this study showed that bioluminescence imaging is a powerful and quantitative tool for the direct assay to monitor tumor growth in vivo. The dual reporter genes transfected tumor-bearing animal model can be applied in the evaluation of the efficacy of new developed anti-cancer drugs

  12. Synovial tissue hypoxia and inflammation in vivo.

    LENUS (Irish Health Repository)

    Ng, C T

    2012-02-01

    INTRODUCTION: Hypoxia is a microenvironmental feature in the inflamed joint, which promotes survival advantage for cells. The aim of this study was to examine the relationship of partial oxygen pressure in the synovial tissue (tPO(2)) in patients with inflammatory arthritis with macroscopic\\/microscopic inflammation and local levels of proinflammatory mediators. METHODS: Patients with inflammatory arthritis underwent full clinical assessment and video arthroscopy to quantify macroscopic synovitis and measure synovial tPO(2) under direct visualisation. Cell specific markers (CD3 (T cells), CD68 (macrophages), Ki67 (cell proliferation) and terminal deoxynucleotidyl transferase dUTP nick end labelling (cell apoptosis)) were quantified by immunohistology. In vitro migration was assessed in primary and normal synoviocytes (synovial fibroblast cells (SFCs)) using a wound repair scratch assay. Levels of tumour necrosis factor alpha (TNFalpha), interleukin 1beta (IL1beta), interferon gamma (IFNgamma), IL6, macrophage inflammatory protein 3alpha (MIP3alpha) and IL8 were quantified, in matched serum and synovial fluid, by multiplex cytokine assay and ELISA. RESULTS: The tPO(2) was 22.5 (range 3.2-54.1) mm Hg and correlated inversely with macroscopic synovitis (r=-0.421, p=0.02), sublining CD3 cells (-0.611, p<0.01) and sublining CD68 cells (r=-0.615, p<0.001). No relationship with cell proliferation or apoptosis was found. Primary and normal SFCs exposed to 1% and 3% oxygen (reflecting the median tPO(2) in vivo) induced cell migration. This was coupled with significantly higher levels of synovial fluid tumour necrosis factor alpha (TNFalpha), IL1beta, IFNgamma and MIP3alpha in patients with tPO(2) <20 mm Hg (all p values <0.05). CONCLUSIONS: This is the first study to show a direct in vivo correlation between synovial tPO(2), inflammation and cell migration, thus it is proposed that hypoxia is a possible primary driver of inflammatory processes in the arthritic joint.

  13. Drosophila melanogaster as an animal model for the study of Pseudomonas aeruginosa biofilm infections in vivo.

    Directory of Open Access Journals (Sweden)

    Heidi Mulcahy

    2011-10-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in susceptible hosts. Chronic P. aeruginosa infections are thought to be caused by bacterial biofilms. Biofilms are highly structured, multicellular, microbial communities encased in an extracellular matrix that enable long-term survival in the host. The aim of this research was to develop an animal model that would allow an in vivo study of P. aeruginosa biofilm infections in a Drosophila melanogaster host. At 24 h post oral infection of Drosophila, P. aeruginosa biofilms localized to and were visualized in dissected Drosophila crops. These biofilms had a characteristic aggregate structure and an extracellular matrix composed of DNA and exopolysaccharide. P. aeruginosa cells recovered from in vivo grown biofilms had increased antibiotic resistance relative to planktonically grown cells. In vivo, biofilm formation was dependent on expression of the pel exopolysaccharide genes, as a pelB::lux mutant failed to form biofilms. The pelB::lux mutant was significantly more virulent than PAO1, while a hyperbiofilm strain (PAZHI3 demonstrated significantly less virulence than PAO1, as indicated by survival of infected flies at day 14 postinfection. Biofilm formation, by strains PAO1 and PAZHI3, in the crop was associated with induction of diptericin, cecropin A1 and drosomycin antimicrobial peptide gene expression 24 h postinfection. In contrast, infection with the non-biofilm forming strain pelB::lux resulted in decreased AMP gene expression in the fly. In summary, these results provide novel insights into host-pathogen interactions during P. aeruginosa oral infection of Drosophila and highlight the use of Drosophila as an infection model that permits the study of P. aeruginosa biofilms in vivo.

  14. [Comparative mutagenesis of human cells in vivo and in vitro]. Progress report, January 1-December 30, 1985

    International Nuclear Information System (INIS)

    1985-01-01

    Annual progress report is made on project focusing on the comparative mutagenesis of human cells in vivo and in vitro. The study employs the HGPRT gene to explore the changes in nucleotide sequence which has occurred in spontaneous mutations or mutations induced by MNNG or ICR191. Reports on the individual projects have been abstracted and indexed for the Energy Data Base. (DT)

  15. MOSFET dosimetry: temperature effects in-vivo

    International Nuclear Information System (INIS)

    Yu, P.K.N.; Cheung, T.; Butson, M.J.; Cancer Services, Wollongong, NSW

    2004-01-01

    Full text: This note investigates temperature effects on dosimetry using a Metal Oxide Semiconductor Field Effect Transistor (MOSFET) for radiotherapy x-ray treatment. This was performed by analysing the dose response and threshold voltage outputs for MOSFET dosimeters as a function of ambient temperature. Results have shown the clinical semiconductor dosimetry system (CSDS) MOSFET provides stable dose measurements with temperatures varying from 15 deg C up to 40 deg C. Thus standard irradiations performed at room temperature can be directly compared to in-vivo dose assessments performed at near body temperature without a temperature correction function. The MOSFET dosimeter threshold voltage varies with temperature and this level is dependant on the dose history of the MOSFET dosimeter. However the variation can be accounted for in the measurement method. For accurate dosimetry the detector should be placed for approximately 60 seconds on a patient to allow thermal equilibrium before measurements are taken with the final reading performed whilst still attached to the patient or conversely left for approximately 120 seconds after removal from the patient if initial readout was measured at room temperature to allow temperature equilibrium to be established. Copyright (2004) Australasian College of Physical Scientists and Engineers in Medicine

  16. Lymphocyte electrotaxis in vitro and in vivo.

    Science.gov (United States)

    Lin, Francis; Baldessari, Fabio; Gyenge, Christina Crenguta; Sato, Tohru; Chambers, Robert D; Santiago, Juan G; Butcher, Eugene C

    2008-08-15

    Electric fields are generated in vivo in a variety of physiologic and pathologic settings, including penetrating injury to epithelial barriers. An applied electric field with strength within the physiologic range can induce directional cell migration (i.e., electrotaxis) of epithelial cells, endothelial cells, fibroblasts, and neutrophils suggesting a potential role in cell positioning during wound healing. In the present study, we investigated the ability of lymphocytes to respond to applied direct current (DC) electric fields. Using a modified Transwell assay and a simple microfluidic device, we show that human PBLs migrate toward the cathode in physiologically relevant DC electric fields. Additionally, electrical stimulation activates intracellular kinase signaling pathways shared with chemotactic stimuli. Finally, video microscopic tracing of GFP-tagged immunocytes in the skin of mouse ears reveals that motile cutaneous T cells actively migrate toward the cathode of an applied DC electric field. Lymphocyte positioning within tissues can thus be manipulated by externally applied electric fields, and may be influenced by endogenous electrical potential gradients as well.

  17. Radical reactions in vivo - an overview

    International Nuclear Information System (INIS)

    Saran, M.; Bors, W.

    1990-01-01

    Generation of radicals in vivo depends on metabolic activities. The reactions are usually influenced by (i) the presence and concentration of oxygen, (ii) the availability of transition metals (effects of binding and compartimentalization), (iii) the level of reductants and antioxidants (e.g. nutritional effects). The effects of radicals are thought to be due to (i) membrane damage (affecting passive or active transport through altered fluidity/function interrelationships, intercellular messenging through modifications in the synthesis of prostaglandins and leukotrienes); (ii) protein damage (e.g. affecting membrane transporters, channel proteins, receptor or regulatory proteins, immunomodulators); (iii) damage to DNA. Defense mechanisms consist of (i) prevention of the 'spreading' of primary damage by low molecular weight antioxidants (e.g. vitamin E, GSH, vitamin C, β-carotene, uric acid); (ii) prevention or limitation of 'secondary' damage by enzymes (e.g. GSH-peroxidase, catalase, superoxide dismutase, DT-diaphorase) and/or chelators; (iii) repair processes, e.g. lipid degradation/membrane repair enzymes (phospholipases, peroxidases, some transferases and reductases), protein disposal or repair enzymes (proteases, GSSG-reductase), DNA degradation or repair enzymes (exonucleases III, endonucleases III and IV, glycosylases, polymerases). Recent hypotheses on a messenging function of the superoxide anion O 2 - are discussed and possible implications of cross-reactions between O 2 - and nitric oxide (endothelium-derived relaxing factor EDRF) are shortly mentioned. (orig.)

  18. In vivo photoacoustic imaging of mouse embryos

    Science.gov (United States)

    Laufer, Jan; Norris, Francesca; Cleary, Jon; Zhang, Edward; Treeby, Bradley; Cox, Ben; Johnson, Peter; Scambler, Pete; Lythgoe, Mark; Beard, Paul

    2012-06-01

    The ability to noninvasively image embryonic vascular anatomy in mouse models is an important requirement for characterizing the development of the normal cardiovascular system and malformations in the heart and vascular supply. Photoacoustic imaging, which can provide high resolution non invasive images of the vasculature based upon optical absorption by endogenous hemoglobin, is well suited to this application. In this study, photoacoustic images of mouse embryos were obtained ex vivo and in vivo. The images show intricate details of the embryonic vascular system to depths of up to 10 mm, which allowed whole embryos to be imaged in situ. To achieve this, an all-optical photoacoustic scanner and a novel time reversal image reconstruction algorithm, which provide deep tissue imaging capability while maintaining high spatial resolution and contrast were employed. This technology may find application as an imaging tool for preclinical embryo studies in developmental biology as well as more generally in preclinical and clinical medicine for studying pathologies characterized by changes in the vasculature.

  19. Optical stimulation of peripheral nerves in vivo

    Science.gov (United States)

    Wells, Jonathon D.

    This dissertation documents the emergence and validation of a new clinical tool that bridges the fields of biomedical optics and neuroscience. The research herein describes an innovative method for direct neurostimulation with pulsed infrared laser light. Safety and effectiveness of this technique are first demonstrated through functional stimulation of the rat sciatic nerve in vivo. The Holmium:YAG laser (lambda = 2.12 mum) is shown to operate at an optimal wavelength for peripheral nerve stimulation with advantages over standard electrical neural stimulation; including contact-free stimulation, high spatial selectivity, and lack of a stimulation artifact. The underlying biophysical mechanism responsible for transient optical nerve stimulation appears to be a small, absorption driven thermal gradient sustained at the axonal layer of nerve. Results explicitly prove that low frequency optical stimulation can reliably stimulate without resulting in tissue thermal damage. Based on the positive results from animal studies, these optimal laser parameters were utilized to move this research into the clinic with a combined safety and efficacy study in human subjects undergoing selective dorsal rhizotomy. The clinical Holmium:YAG laser was used to effectively stimulate human dorsal spinal roots and elicit functional muscle responses recorded during surgery without evidence of nerve damage. Overall these results predict that this technology can be a valuable clinical tool in various neurosurgical applications.

  20. Differential in vivo regulation of TH and DBH mRNA in rat atria by maprotiline and fluoxetine

    Directory of Open Access Journals (Sweden)

    Spasojević Nataša

    2011-01-01

    Full Text Available It is well known that antidepressants affect central monoaminergic neurotransmission and that they also modulate hormone release in peripheral tissues. Repeated maprotiline (a noradrenaline reuptake inhibitor and fluoxetine (a serotonin reuptake inhibitor treatment on gene expression of the catecholamine biosynthetic enzymes were examined in rat atria and ventricles in vivo. Maprotiline decreased the gene expression of tyrosine hydroxylase (TH and dopamineβ-hydroxylase (DBH in the rat atrium. Fluoxetine increased gene expression of TH and DBH, but not of phenylethanolamine N-methyltransferase (PNMT. Chronic application of antidepressants did not change the expression of these enzymes in the ventricles. We conclude that repeated administration of fluoxetine enhances gene transcription of TH and DBH and subsequently stimulates noradrenaline synthesis in rat atria in vivo.

  1. In vitro and in vivo Development of Cloned Ovine Embryos using in vitro and in vivo Matured Oocytes

    DEFF Research Database (Denmark)

    Holm, P; Nagashima, H; Sun, F-J

    1995-01-01

    Cloning of sheep embryos by nucleus transplantation can be achieved by using in vivo matured (oviductal) oocytes and in vivo culture. However, these steps involve cumbersome procedures. Therefore, the effects of in vivo vs. the equivalent in vitro procedures on the pre-implantation development...... matured oocytes were enucleated and fused with inserted blastomeres from donor embryos. In vitro matured oocytes were enucleated and allowed to age prior to blastomere insertion and electrofusion. Fused embryos were cultured for approximately 132 h either in vivo in ligated sheep oviducts or in vitro...

  2. Synthetic biology devices for in vitro and in vivo diagnostics.

    Science.gov (United States)

    Slomovic, Shimyn; Pardee, Keith; Collins, James J

    2015-11-24

    There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms.

  3. Regenerative orthopaedics: in vitro, in vivo...in silico.

    Science.gov (United States)

    Geris, Liesbet

    2014-09-01

    In silico, defined in analogy to in vitro and in vivo as those studies that are performed on a computer, is an essential step in problem-solving and product development in classical engineering fields. The use of in silico models is now slowly easing its way into medicine. In silico models are already used in orthopaedics for the planning of complicated surgeries, personalised implant design and the analysis of gait measurements. However, these in silico models often lack the simulation of the response of the biological system over time. In silico models focusing on the response of the biological systems are in full development. This review starts with an introduction into in silico models of orthopaedic processes. Special attention is paid to the classification of models according to their spatiotemporal scale (gene/protein to population) and the information they were built on (data vs hypotheses). Subsequently, the review focuses on the in silico models used in regenerative orthopaedics research. Contributions of in silico models to an enhanced understanding and optimisation of four key elements-cells, carriers, culture and clinics-are illustrated. Finally, a number of challenges are identified, related to the computational aspects but also to the integration of in silico tools into clinical practice.

  4. Atm reactivation reverses ataxia telangiectasia phenotypes in vivo.

    Science.gov (United States)

    Di Siena, Sara; Campolo, Federica; Gimmelli, Roberto; Di Pietro, Chiara; Marazziti, Daniela; Dolci, Susanna; Lenzi, Andrea; Nussenzweig, Andre; Pellegrini, Manuela

    2018-02-22

    Hereditary deficiencies in DNA damage signaling are invariably associated with cancer predisposition, immunodeficiency, radiation sensitivity, gonadal abnormalities, premature aging, and tissue degeneration. ATM kinase has been established as a central player in DNA double-strand break repair and its deficiency causes ataxia telangiectasia, a rare, multi-system disease with no cure. So ATM represents a highly attractive target for the development of novel types of gene therapy or transplantation strategies. Atm tamoxifen-inducible mouse models were generated to explore whether Atm reconstitution is able to restore Atm function in an Atm-deficient background. Body weight, immunodeficiency, spermatogenesis, and radioresistance were recovered in transgenic mice within 1 month from Atm induction. Notably, life span was doubled after Atm restoration, mice were protected from thymoma and no cerebellar defects were observed. Atm signaling was functional after DNA damage in vivo and in vitro. In summary, we propose a new Atm mouse model to investigate novel therapeutic strategies for ATM activation in ataxia telangiectasia disease.

  5. In vivo cardiac role of migfilin during experimental pressure overload.

    Science.gov (United States)

    Haubner, Bernhard Johannes; Moik, Daniel; Schuetz, Thomas; Reiner, Martin F; Voelkl, Jakob G; Streil, Katrin; Bader, Kerstin; Zhao, Lei; Scheu, Claudia; Mair, Johannes; Pachinger, Otmar; Metzler, Bernhard

    2015-06-01

    Increased myocardial wall strain triggers the cardiac hypertrophic response by increasing cardiomyocyte size, reprogramming gene expression, and enhancing contractile protein synthesis. The LIM protein, migfilin, is a cytoskeleton-associated protein that was found to translocate in vitro into the nucleus in a Ca(2+)-dependent manner, where it co-activates the pivotal cardiac transcription factor Csx/Nkx2.5. However, the in vivo role of migfilin in cardiac function and stress response is unclear. To define the role of migfilin in cardiac hypertrophy, we induced hypertension by transverse aortic constriction (TAC) and compared cardiac morphology and function of migfilin knockout (KO) with wild-type (WT) hearts. Heart size and myocardial contractility were comparable in untreated migfilin KO and WT hearts, but migfilin-null hearts presented a reduced extent of hypertrophic remodelling in response to chronic hypertensile stress. Migfilin KO mice maintained their cardiac function for a longer time period compared with WT mice, which presented extensive fibrosis and death due to heart failure. Migfilin translocated into the nucleus of TAC-treated cardiomyocytes, and migfilin KO hearts showed reduced Akt activation during the early response to pressure overload. Our findings indicate an important role of migfilin in the regulation of cardiac hypertrophy upon experimental TAC. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

  6. Uncaria rhynchophylla induces angiogenesis in vitro and in vivo.

    Science.gov (United States)

    Choi, Do-Young; Huh, Jeong-Eun; Lee, Jae-Dong; Cho, Eun-Mi; Baek, Yong-Hyeon; Yang, Ha-Ru; Cho, Yoon-Je; Kim, Kang-Il; Kim, Deog-Yoon; Park, Dong-Suk

    2005-12-01

    Angiogenesis consists of the proliferation, migration, and differentiation of endothelial cells, and angiogenic factors and matrix protein interactions modulate this process. The aim of this study was to determine the angiogenic properties of Uncaria rhynchophylla. Uncaria rhynchophylla significantly enhanced human umbilical vein endothelial cells (HUVECs) proliferation in a dose-dependent manner. Neutralization of vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) by monoclonal antibody suppressed the Uncaria rhynchophylla stimulatory effect on proliferation. In addition, Uncaria rhynchophylla significantly increased chemotactic-migration on gelatin and tubular structures on Matrigel of HUVECs in a dose-dependent manner. Interestingly, Uncaria rhynchophylla dose-dependently increased VEGF, and bFGF gene expression and protein secretion of HUVEC. The angiogenic activity of Uncaria rhynchophylla was confirmed using an in vivo Matrigel angiogenesis model, showing promotion of blood vessel formation. These results suggest that Uncaria rhynchophylla could potentially used to accelerate vascular wound healing or to promote the growth of collateral blood vessel in ischemic tissues.

  7. In Vivo Enhancer Analysis Chromosome 16 Conserved NoncodingSequences

    Energy Technology Data Exchange (ETDEWEB)

    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 Characterization of Human APOA5 Haplotypes

    Energy Technology Data Exchange (ETDEWEB)

    Ahituv, Nadav; Akiyama, Jennifer; Chapman-Helleboid, Audrey; Fruchart, Jamila; Pennacchio, Len A.

    2006-10-01

    Increased plasma triglycerides concentrations are an independent risk factor for cardiovascular disease. Numerous studies support a reproducible genetic association between two minor haplotypes in the human apolipoprotein A5 gene (APOA5) and increased plasma triglyceride concentrations. We thus sought to investigate the effect of these minor haplotypes (APOA5*2 and APOA5*3) on ApoAV plasma levels through the precise insertion of single-copy intact APOA5 haplotypes at a targeted location in the mouse genome. While we found no difference in the amount of human plasma ApoAV in mice containing the common APOA5*1 and minor APOA5*2 haplotype, the introduction of the single APOA5*3 defining allele (19W) resulted in 3-fold lower ApoAV plasma levels consistent with existing genetic association studies. These results indicate that S19W polymorphism is likely to be functional and explain the strong association of this variant with plasma triglycerides supporting the value of sensitive in vivo assays to define the functional nature of human haplotypes.

  9. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection.

    Science.gov (United States)

    Bikard, David; Hatoum-Aslan, Asma; Mucida, Daniel; Marraffini, Luciano A

    2012-08-16

    Pathogenic bacterial strains emerge largely due to transfer of virulence and antimicrobial resistance genes between bacteria, a process known as horizontal gene transfer (HGT). Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci of bacteria and archaea encode a sequence-specific defense mechanism against bacteriophages and constitute a programmable barrier to HGT. However, the impact of CRISPRs on the emergence of virulence is unknown. We programmed the human pathogen Streptococcus pneumoniae with CRISPR sequences that target capsule genes, an essential pneumococcal virulence factor, and show that CRISPR interference can prevent transformation of nonencapsulated, avirulent pneumococci into capsulated, virulent strains during infection in mice. Further, at low frequencies bacteria can lose CRISPR function, acquire capsule genes, and mount a successful infection. These results demonstrate that CRISPR interference can prevent the emergence of virulence in vivo and that strong selective pressure for virulence or antibiotic resistance can lead to CRISPR loss in bacterial pathogens. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Measuring protein breakdown rate in individual proteins in vivo

    DEFF Research Database (Denmark)

    Holm, Lars; Kjaer, Michael

    2010-01-01

    To outline different approaches of how protein breakdown can be quantified and to present a new approach to determine the fractional breakdown rate of individual slow turnover proteins in vivo.......To outline different approaches of how protein breakdown can be quantified and to present a new approach to determine the fractional breakdown rate of individual slow turnover proteins in vivo....

  11. In-vivo evaluation of convex array synthetic aperture imaging

    DEFF Research Database (Denmark)

    Pedersen, Morten Høgholm; Gammelmark, Kim Løkke; Jensen, Jørgen Arendt

    2007-01-01

    This paper presents an in-vivo study of synthetic transmit aperture (STA) imaging in comparison to conventional imaging, evaluating whether STA imaging is feasible in-vivo, and whether the image quality obtained is comparable to traditional scanned imaging in terms of penetration depth, spatial...

  12. Nevomelanocytic atypia detection by in vivo reflectance confocal microscopy

    Directory of Open Access Journals (Sweden)

    Ingrida Vaišnorienė

    2014-01-01

    Conclusions: Nevus with histopathologically confirmed nevomelanocytic atypia (dysplastic nevus could not be distinguished from nevus without atypia using analyzed in vivo RCM features of melanocytic atypia. More accurate diagnostics by means of in vivo RCM needs further investigation on reflectance of single and nested cutaneous melanocytes in benign and malignant skin lesions.

  13. In vivo monitoring of angiogenesis within Matrigel chambers using MRI

    DEFF Research Database (Denmark)

    Holm, David; Ley, Carsten Dan; Søgaard, Lise Vejby

    2006-01-01

    Angiogenesis is a critical process in tumour development and presents an important target for the development of a range of anti-cancer agents . To assess the in vivo efficacy of these ‘angiotherapeutics', a simple and reproducible in vivo model would be of significant value. Here we show...

  14. Thyroid endocrine system disruption by pentachlorophenol: an in vitro and in vivo assay.

    Science.gov (United States)

    Guo, Yongyong; Zhou, Bingsheng

    2013-10-15

    The present study aimed to evaluate the disruption caused to the thyroid endocrine system by pentachlorophenol (PCP) using in vitro and in vivo assays. In the in vitro assay, rat pituitary GH3 cells were exposed to 0, 0.1, 0.3, and 1.0 μM PCP. PCP exposure significantly downregulated basal and triiodothyronine (T3)-induced Dio 1 transcription, indicating the antagonistic activity of PCP in vitro. In the in vivo assay, zebrafish embryos were exposed to 0, 1, 3, and 10 μg/L of PCP until 14 days post-fertilization. PCP exposure resulted in decreased thyroxine (T4) levels, but elevated contents of whole-body T3. PCP exposure significantly upregulated the mRNA expression of genes along hypothalamic-pituitary-thyroid (HPT) axis, including those encoding thyroid-stimulating hormone, sodium/iodide symporter, thyroglobulin, Dio 1 and Dio 2, alpha and beta thyroid hormone receptor, and uridinediphosphate-glucuronosyl-transferase. PCP exposure did not influence the transcription of the transthyretin (TTR) gene. The results indicate that PCP potentially disrupts the thyroid endocrine system both in vitro and in vivo. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. In vivo therapeutic responses contingent on Fanconi anemia/BRCA2 status of the tumor.

    Science.gov (United States)

    van der Heijden, Michiel S; Brody, Jonathan R; Dezentje, David A; Gallmeier, Eike; Cunningham, Steven C; Swartz, Michael J; DeMarzo, Angelo M; Offerhaus, G Johan A; Isacoff, William H; Hruban, Ralph H; Kern, Scott E

    2005-10-15

    BRCA2, FANCC, and FANCG gene mutations are present in a subset of pancreatic cancer. Defects in these genes could lead to hypersensitivity to interstrand cross-linkers in vivo and a more optimal treatment of pancreatic cancer patients based on the genetic profile of the tumor. Two retrovirally complemented pancreatic cancer cell lines having defects in the Fanconi anemia pathway, PL11 (FANCC-mutated) and Hs766T (FANCG-mutated), as well as several parental pancreatic cancer cell lines with or without mutations in the Fanconi anemia/BRCA2 pathway, were assayed for in vitro and in vivo sensitivities to various chemotherapeutic agents. A distinct dichotomy of drug responses was observed. Fanconi anemia-defective cancer cells were hypersensitive to the cross-linking agents mitomycin C (MMC), cisplatin, chlorambucil, and melphalan but not to 5-fluorouracil, gemcitabine, doxorubicin, etoposide, vinblastine, or paclitaxel. Hypersensitivity to cross-linking agents was confirmed in vivo; FANCC-deficient xenografts of PL11 and BRCA2-deficient xenografts of CAPAN1 regressed on treatment with two different regimens of MMC whereas Fanconi anemia-proficient xenografts did not. The MMC response comprised cell cycle arrest, apoptosis, and necrosis. Xenografts of PL11 also regressed after a single dose of cyclophosphamide whereas xenografts of genetically complemented PL11(FANCC) did not. MMC or other cross-linking agents as a clinical therapy for pancreatic cancer patients with tumors harboring defects in the Fanconi anemia/BRCA2 pathway should be specifically investigated.

  16. Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo

    Directory of Open Access Journals (Sweden)

    Jing Zhou

    2017-12-01

    Full Text Available Far-red fluorescent reporter genes can be used for tracking cells non-invasively in vivo using fluorescence imaging. Here, we investigate the effectiveness of the far-red fluorescent protein, E2-Crimson (E2C, for tracking mouse embryonic cells (mESCs in vivo following subcutaneous administration into mice. Using a knock-in strategy, we introduced E2C into the Rosa26 locus of an E14-Bra-GFP mESC line, and after confirming that the E2C had no obvious effect on the phenotype of the mESCs, we injected them into mice and imaged them over nine days. The results showed that fluorescence intensity was weak, and cells could only be detected when injected at high densities. Furthermore, intensity peaked on day 4 and then started to decrease, despite the fact that tumour volume continued to increase beyond day 4. Histopathological analysis showed that although E2C fluorescence could barely be detected in vivo at day 9, analysis of frozen sections indicated that all mESCs within the tumours continued to express E2C. We hypothesise that the decrease in fluorescence intensity in vivo was probably due to the fact that the mESC tumours became more vascular with time, thus leading to increased absorbance of E2C fluorescence by haemoglobin. We conclude that the E2C reporter has limited use for tracking cells in vivo, at least when introduced as a single copy into the Rosa26 locus.

  17. In Vivo Imaging of Human Malignant Mesothelioma Grown Orthotopically in the Peritoneal Cavity of Nude Mice

    Directory of Open Access Journals (Sweden)

    Mingqian Feng, Jingli Zhang, Miriam Anver, Raffit Hassan, Mitchell Ho

    2011-01-01

    Full Text Available Malignant mesothelioma (MM causes significant morbidity and mortality in patients. With increasing efforts devoted to developing therapeutics targeting mesothelioma, a xenograft mouse model with in vivo tumor imaging is especially desired for evaluating anti-tumor therapies. In the present study, we fluorescently labeled the NCI-H226 human mesothelioma cell line by a lentiviral vector harboring a luciferase-GFP (Luc/GFP fusion gene driven by the RNA polymerase II promoter. After single-cell cloning by flow cytometry, a clone (named LMB-H226-GL that stably expresses high levels of Luc/GFP was obtained. The in vivo tumorigenicity of Luc/GFP-labeled LMB-H226-GL was determined by using intraperitoneal injections of the cells in nude mice. LMB-H226-GL was found to be able to consistently form solid tumors in the peritoneum of mice. Tumor growth and aggressive progression could be quantitated via in vivo bioluminescence imaging. The model exhibited the pathological hallmarks consistent with the clinical progression of MM in terms of tumor growth and spread inside the peritoneal cavity. To evaluate the in vivo efficacy of drugs targeting mesothelioma, we treated mice with SS1P, a recombinant immunotoxin currently evaluated in Phase II clinical trials for treatment of mesothelioma. All the tumor-bearing mice had a significant response to SS1P treatment. Our results showed that this is a well-suited model for mesothelioma, and may be useful for evaluating other novel agents for mesothelioma treatment in vivo.

  18. Transcriptional regulation of rod photoreceptor homeostasis revealed by in vivo NRL targetome analysis.

    Directory of Open Access Journals (Sweden)

    Hong Hao

    Full Text Available 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.

  19. Genetic Determinants Associated With in Vivo Survival of Burkholderia cenocepacia in the Caenorhabditis elegans Model

    KAUST Repository

    Wong, Yee-Chin

    2018-05-29

    A Burkholderia cenocepacia infection usually leads to reduced survival and fatal cepacia syndrome in cystic fibrosis patients. The identification of B. cenocepacia essential genes for in vivo survival is key to designing new anti-infectives therapies. We used the Transposon-Directed Insertion Sequencing (TraDIS) approach to identify genes required for B. cenocepacia survival in the model infection host, Caenorhabditis elegans. A B. cenocepacia J2315 transposon pool of ∼500,000 mutants was used to infect C. elegans. We identified 178 genes as crucial for B. cenocepacia survival in the infected nematode. The majority of these genes code for proteins of unknown function, many of which are encoded by the genomic island BcenGI13, while other gene products are involved in nutrient acquisition, general stress responses and LPS O-antigen biosynthesis. Deletion of the glycosyltransferase gene wbxB and a histone-like nucleoid structuring (H-NS) protein-encoding gene (BCAL0154) reduced bacterial accumulation and attenuated virulence in C. elegans. Further analysis using quantitative RT-PCR indicated that BCAL0154 modulates B. cenocepacia pathogenesis via transcriptional regulation of motility-associated genes including fliC, fliG, flhD, and cheB1. This screen has successfully identified genes required for B. cenocepacia survival within the host-associated environment, many of which are potential targets for developing new antimicrobials.

  20. Genetic Determinants Associated With in Vivo Survival of Burkholderia cenocepacia in the Caenorhabditis elegans Model

    KAUST Repository

    Wong, Yee-Chin; Abd El Ghany, Moataz; Ghazzali, Raeece N. M.; Yap, Soon-Joo; Hoh, Chee-Choong; Pain, Arnab; Nathan, Sheila

    2018-01-01

    A Burkholderia cenocepacia infection usually leads to reduced survival and fatal cepacia syndrome in cystic fibrosis patients. The identification of B. cenocepacia essential genes for in vivo survival is key to designing new anti-infectives therapies. We used the Transposon-Directed Insertion Sequencing (TraDIS) approach to identify genes required for B. cenocepacia survival in the model infection host, Caenorhabditis elegans. A B. cenocepacia J2315 transposon pool of ∼500,000 mutants was used to infect C. elegans. We identified 178 genes as crucial for B. cenocepacia survival in the infected nematode. The majority of these genes code for proteins of unknown function, many of which are encoded by the genomic island BcenGI13, while other gene products are involved in nutrient acquisition, general stress responses and LPS O-antigen biosynthesis. Deletion of the glycosyltransferase gene wbxB and a histone-like nucleoid structuring (H-NS) protein-encoding gene (BCAL0154) reduced bacterial accumulation and attenuated virulence in C. elegans. Further analysis using quantitative RT-PCR indicated that BCAL0154 modulates B. cenocepacia pathogenesis via transcriptional regulation of motility-associated genes including fliC, fliG, flhD, and cheB1. This screen has successfully identified genes required for B. cenocepacia survival within the host-associated environment, many of which are potential targets for developing new antimicrobials.

  1. In vivo NMR spectroscopy of the liver. Spectroscopie RMN du foie in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Jehenson, P.; Cuenod, C.A.; Syrota, A. (CEA, 91 - Orsay (FR). Service Hospitalier Frederic Joliot)

    1989-01-01

    The application of in vivo MR spectroscopy to the study of the liver is currently an expanding field of research. Owing to technical difficulties, the results obtained thus far were mainly those of animal observations. Several nuclei have been considered: hydrogen, phosphorus, carbon or fluorine. This non-traumatic method allows following and quantifying the various metabolic pathways, especially during hepatic diseases. The major metabolic pathways, i.e. neoglycogenesis, glycogenolysis, Krebs' cycle, etc., are studied, as well as their alterations during diseases such as ischemia, diabetes or alcoholism. The development of this promising technique requires the cooperation of various clinical and fundamental disciplines.

  2. In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

    Science.gov (United States)

    Miller, Peter G.; Al-Shahrour, Fatima; Hartwell, Kimberly A.; Chu, Lisa P.; Järås, Marcus; Puram, Rishi V.; Puissant, Alexandre; Callahan, Kevin P.; Ashton, John; McConkey, Marie E.; Poveromo, Luke P.; Cowley, Glenn S.; Kharas, Michael G.; Labelle, Myriam; Shterental, Sebastian; Fujisaki, Joji; Silberstein, Lev; Alexe, Gabriela; Al-Hajj, Muhammad A.; Shelton, Christopher A.; Armstrong, Scott A.; Root, David E.; Scadden, David T.; Hynes, Richard O.; Mukherjee, Siddhartha; Stegmaier, Kimberly; Jordan, Craig T.; Ebert, Benjamin L.

    2013-01-01

    SUMMARY We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML. PMID:23770013

  3. Loss of Optineurin In Vivo Results in Elevated Cell Death and Alters Axonal Trafficking Dynamics

    Science.gov (United States)

    Paulus, Jeremiah D.; Link, Brian A.

    2014-01-01

    Mutations in Optineurin have been associated with ALS, glaucoma, and Paget’s disease of bone in humans, but little is known about how these mutations contribute to disease. Most of the cellular consequences of Optineurin loss have come from in vitro studies, and it remains unclear whether these same defects would be seen in vivo. To answer this question, we assessed the cellular consequences of Optineurin loss in zebrafish embryos to determine if they showed the same defects as have been described in the in vitro studies. We found that loss of Optineurin resulted in increased cell death, as well as subtle cell morphology, cell migration and vesicle trafficking defects. However, unlike experiments on cells in culture, we found no indication that the Golgi apparatus was disrupted or that NF-κB target genes were upregulated. Therefore, we conclude that in vivo loss of Optineurin shows some, but not all, of the defects seen in in vitro work. PMID:25329564

  4. In vivo differentiation of induced pluripotent stem cells into neural stem cells by chimera formation.

    Science.gov (United States)

    Choi, Hyun Woo; Hong, Yean Ju; Kim, Jong Soo; Song, Hyuk; Cho, Ssang Gu; Bae, Hojae; Kim, Changsung; Byun, Sung June; Do, Jeong Tae

    2017-01-01

    Like embryonic stem cells, induced pluripotent stem cells (iPSCs) can differentiate into all three germ layers in an in vitro system. Here, we developed a new technology for obtaining neural stem cells (NSCs) from iPSCs through chimera formation, in an in vivo environment. iPSCs contributed to the neural lineage in the chimera, which could be efficiently purified and directly cultured as NSCs in vitro. The iPSC-derived, in vivo-differentiated NSCs expressed NSC markers, and their gene-expression pattern more closely resembled that of fetal brain-derived NSCs than in vitro-differentiated NSCs. This system could be applied for differentiating pluripotent stem cells into specialized cell types whose differentiation protocols are not well established.

  5. In vitro and in vivo screening of azole fungicides for antiandrogenic effects

    DEFF Research Database (Denmark)

    Taxvig, Camilla; Vinggaard, Anne; Hass, Ulla

    signs of feminization of the male offspring were investigated. Tebuconazole caused an increase in testicular 17alfa-hydroxyprogesterone and progesterone levels, and a decrease in testosterone levels in male fetuses. Epoxiconazole had no effect on any of the mesured hormonelevels. Furthermore...... and antiandrogenic effects both in vitro and in vivo. Two other azole fungicides, tebuconazole and epoxiconazole, have now been investigated for antiandrogenic effects in vitro and in vivo as well. The fungicides were screened in two well-established cell assays, including testing for agonistic and antagonistic...... effects on AR in transfected CHO cells, using an AR reporter gene assay. The compounds were also analyzed for effects on steroidogenesis in H295R cells, a human adrenocorticocarcinoma cell line, used to detect effects on steroid production. In vitro tebuconazole and epoxiconazole proved to be antagonists...

  6. In vitro and in vivo hepatoprotective effect of ganodermanontriol against t-BHP-induced oxidative stress

    DEFF Research Database (Denmark)

    Ha, Do Thi; Oh, Joonseok; Khoi, Nguyen Minh

    2013-01-01

    the evaluation of hepatoprotective activity of ganodermanontriol, a sterol isolated from Ganoderma lucidum, and the investigation of its mechanism of action in Hepa1c1c7 and murine liver cells upon tert-butyl hydroperoxide (t-BHP)-induced inflammation. t-BHP was utilized to stimulate an anti...... were monitored to assess whether GDT protected the cells from the t-BHP-mediated oxidative stimuli. RESULTS: GDT induced HO-1 expression via the activation of Nrf-2 nuclear translocation and the subsequent transcription of the HO-1 gene in vitro and in vivo, which seemed to be regulated...... the ethnopharmacological application of Ganoderma lucidum as a treatment for hepatic disorders. GDT induced in vitro and in vivo anti-inflammatory activity in t-BHP-damaged hepatic cells through the expression of HO-1, and in which PI3K/Akt and p38 kinases are involved. Our study motivates further research...

  7. Deducing the kinetics of protein synthesis in vivo from the transition rates measured in vitro.

    Directory of Open Access Journals (Sweden)

    Sophia Rudorf

    2014-10-01

    Full Text Available The molecular machinery of life relies on complex multistep processes that involve numerous individual transitions, such as molecular association and dissociation steps, chemical reactions, and mechanical movements. The corresponding transition rates can be typically measured in vitro but not in vivo. Here, we develop a general method to deduce the in-vivo rates from their in-vitro values. The method has two basic components. First, we introduce the kinetic distance, a new concept by which we can quantitatively compare the kinetics of a multistep process in different environments. The kinetic distance depends logarithmically on the transition rates and can be interpreted in terms of the underlying free energy barriers. Second, we minimize the kinetic distance between the in-vitro and the in-vivo process, imposing the constraint that the deduced rates reproduce a known global property such as the overall in-vivo speed. In order to demonstrate the predictive power of our method, we apply it to protein synthesis by ribosomes, a key process of gene expression. We describe the latter process by a codon-specific Markov model with three reaction pathways, corresponding to the initial binding of cognate, near-cognate, and non-cognate tRNA, for which we determine all individual transition rates in vitro. We then predict the in-vivo rates by the constrained minimization procedure and validate these rates by three independent sets of in-vivo data, obtained for codon-dependent translation speeds, codon-specific translation dynamics, and missense error frequencies. In all cases, we find good agreement between theory and experiment without adjusting any fit parameter. The deduced in-vivo rates lead to smaller error frequencies than the known in-vitro rates, primarily by an improved initial selection of tRNA. The method introduced here is relatively simple from a computational point of view and can be applied to any biomolecular process, for which we have

  8. In Vivo Chromatin Targets of the Transcription Factor Yin Yang 2 in Trophoblast Stem Cells

    Science.gov (United States)

    Pérez-Palacios, Raquel; Macías-Redondo, Sofía; Climent, María; Contreras-Moreira, Bruno; Muniesa, Pedro; Schoorlemmer, Jon

    2016-01-01

    Background Yin Yang 2 (YY2) is a zinc finger protein closely related to the well-characterized Yin Yang 1 (YY1). YY1 is a DNA-binding transcription factor, with defined functions in multiple developmental processes, such as implantation, cell differentiation, X inactivation, imprinting and organogenesis. Yy2 has been treated as a largely immaterial duplication of Yy1, as they share high homology in the Zinc Finger-region and similar if not identical in vitro binding sites. In contrast to these similarities, gene expression alterations in HeLa cells with attenuated levels of either Yy1 or Yy2 were to some extent gene-specific. Moreover, the chromatin binding sites for YY2, except for its association with transposable retroviral elements (RE) and Endogenous Retroviral Elements (ERVs), remain to be identified. As a first step towards defining potential Yy2 functions matching or complementary to Yy1, we considered in vivo DNA binding sites of YY2 in trophoblast stem (TS) cells. Results We report the presence of YY2 protein in mouse-derived embryonic stem (ES) and TS cell lines. Following up on our previous report on ERV binding by YY2 in TS cells, we investigated the tissue-specificity of REX1 and YY2 binding and confirm binding to RE/ERV targets in both ES cells and TS cells. Because of the higher levels of expression, we chose TS cells to understand the role of Yy2 in gene and chromatin regulation. We used in vivo YY2 association as a measure to identify potential target genes. Sequencing of chromatin obtained in chromatin-immunoprecipitation (ChIP) assays carried out with αYY2 serum allowed us to identify a limited number of chromatin targets for YY2. Some putative binding sites were validated in regular ChIP assays and gene expression of genes nearby was altered in the absence of Yy2. Conclusions YY2 binding to ERVs is not confined to TS cells. In vivo binding sites share the presence of a consensus binding motif. Selected sites were uniquely bound by YY2 as

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

    Science.gov (United States)

    Attridge, Kesley; Walker, Lucy S K

    2014-01-01

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

  10. In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features.

    Science.gov (United States)

    Ding, Yiliang; Tang, Yin; Kwok, Chun Kit; Zhang, Yu; Bevilacqua, Philip C; Assmann, Sarah M

    2014-01-30

    RNA structure has critical roles in processes ranging from ligand sensing to the regulation of translation, polyadenylation and splicing. However, a lack of genome-wide in vivo RNA structural data has limited our understanding of how RNA structure regulates gene expression in living cells. Here we present a high-throughput, genome-wide in vivo RNA structure probing method, structure-seq, in which dimethyl sulphate methylation of unprotected adenines and cytosines is identified by next-generation sequencing. Application of this method to Arabidopsis thaliana seedlings yielded the first in vivo genome-wide RNA structure map at nucleotide resolution for any organism, with quantitative structural information across more than 10,000 transcripts. Our analysis reveals a three-nucleotide periodic repeat pattern in the structure of coding regions, as well as a less-structured region immediately upstream of the start codon, and shows that these features are strongly correlated with translation efficiency. We also find patterns of strong and weak secondary structure at sites of alternative polyadenylation, as well as strong secondary structure at 5' splice sites that correlates with unspliced events. Notably, in vivo structures of messenger RNAs annotated for stress responses are poorly predicted in silico, whereas mRNA structures of genes related to cell function maintenance are well predicted. Global comparison of several structural features between these two categories shows that the mRNAs associated with stress responses tend to have more single-strandedness, longer maximal loop length and higher free energy per nucleotide, features that may allow these RNAs to undergo conformational changes in response to environmental conditions. Structure-seq allows the RNA structurome and its biological roles to be interrogated on a genome-wide scale and should be applicable to any organism.

  11. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

    Science.gov (United States)

    Eskildsen, Tilde; Taipaleenmäki, Hanna; Stenvang, Jan; Abdallah, Basem M.; Ditzel, Nicholas; Nossent, Anne Yael; Bak, Mads; Kauppinen, Sakari; Kassem, Moustapha

    2011-01-01

    Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3′ UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central role in promoting osteoblast differentiation, as a bona fide target of miR-138. We show that miR-138 attenuates bone formation in vivo, at least in part by inhibiting the focal adhesion kinase signaling pathway. Our findings suggest that pharmacological inhibition of miR-138 by antimiR-138 could represent a therapeutic strategy for enhancing bone formation in vivo. PMID:21444814

  12. In vivo cell kinetics in breast carcinogenesis

    International Nuclear Information System (INIS)

    Bai, Maria; Agnantis, Niki J; Kamina, Sevasti; Demou, Asimina; Zagorianakou, Panayiota; Katsaraki, Aphroditi; Kanavaros, Panayiotis

    2001-01-01

    Disruption of the balance between apoptosis and proliferation is considered to be an important factor in the development and progression of tumours. In the present study we determined the in vivo cell kinetics along the spectrum of apparently normal epithelium, hyperplasia, preinvasive lesions and invasive carcinoma, in breast tissues affected by fibrocystic changes in which preinvasive and/or invasive lesions developed, as a model of breast carcinogenesis. A total of 32 areas of apparently normal epithelium and 135 ductal proliferative and neoplastic lesions were studied. More than one epithelial lesion per case were analyzed. The apoptotic index (AI) and the proliferative index (PI) were expressed as the percentage of TdT-mediated dUTP-nick end-labelling (TUNEL) and Ki-67-positive cells, respectively. The PI/AI (P/A index) was calculated for each case. The AIs and PIs were significantly higher in hyperplasia than in apparently normal epithelium (P = 0.04 and P = 0.0005, respectively), in atypical hyperplasia than in hyperplasia (P = 0.01 and P = 0.04, respectively) and in invasive carcinoma than in in situ carcinoma (P < 0.001 and P < 0.001, respectively). The two indices were similar in atypical hyperplasia and in in situ carcinoma. The P/A index increased significantly from normal epithelium to hyperplasia (P = 0.01) and from preinvasive lesions to invasive carcinoma (P = 0.04) whereas it was decreased (non-significantly) from hyperplasia to preinvasive lesions. A strong positive correlation between the AIs and the PIs was found (r = 0.83, P < 0.001). These findings suggest accelerating cell turnover along the continuum of breast carcinogenesis. Atypical hyperplasias and in situ carcinomas might be kinetically similar lesions. In the transition from normal epithelium to hyperplasia and from preinvasive lesions to invasive carcinoma the net growth of epithelial cells results from a growth imbalance in favour of proliferation. In the transition from hyperplasia

  13. Endothelial cells present antigens in vivo

    Directory of Open Access Journals (Sweden)

    Tellides George

    2004-03-01

    Full Text Available Abstract Background Immune recognition of vascular endothelial cells (EC has been implicated in allograft rejection, protection against pathogens, and lymphocyte recruitment. However, EC pervade nearly all tissues and predominate in none, complicating any direct test of immune recognition. Here, we examined antigen presentation by EC in vivo by testing immune responses against E. coli β-galactosidase (β-gal in two lines of transgenic mice that express β-gal exclusively in their EC. TIE2-lacZ mice express β-gal in all EC and VWF-lacZ mice express β-gal in heart and brain microvascular EC. Results Transgenic and congenic wild type FVB mice immunized with β-gal expression vector DNA or β-gal protein generated high titer, high affinity antisera containing comparable levels of antigen-specific IgG1 and IgG2a isotypes, suggesting equivalent activation of T helper cell subsets. The immunized transgenic mice remained healthy, their EC continued to express β-gal, and their blood vessels showed no histological abnormalities. In response to β-gal in vitro, CD4+ and CD8+ T cells from immunized transgenic and FVB mice proliferated, expressed CD25, and secreted IFN-γ. Infection with recombinant vaccinia virus encoding β-gal raised equivalent responses in transgenic and FVB mice. Hearts transplanted from transgenic mice into FVB mice continued to beat and the graft EC continued to express β-gal. These results suggested immunological ignorance of the transgene encoded EC protein. However, skin transplanted from TIE2-lacZ onto FVB mice lost β-gal+ EC and the hosts developed β-gal-specific antisera, demonstrating activation of host immune effector mechanisms. In contrast, skin grafted from TIE2-lacZ onto VWF-lacZ mice retained β-gal+ EC and no antisera developed, suggesting a tolerant host immune system. Conclusion Resting, β-gal+ EC in transgenic mice tolerize specific lymphocytes that would otherwise respond against β-gal expressed by EC within

  14. In vitro-in vivo correlation in skin permeation.

    Science.gov (United States)

    Mohammed, D; Matts, P J; Hadgraft, J; Lane, M E

    2014-02-01

    In vitro skin permeation studies have been used extensively in the development and optimisation of delivery of actives in vivo. However, there are few reported correlations of such in vitro studies with in vivo data. The aim of this study was to investigate the skin permeation of a model active, niacinamide, both in vitro and in vivo. Conventional diffusion cell studies were conducted in human skin to determine niacinamide permeation from a range of vehicles which included dimethyl isosorbide (DMI), propylene glycol (PG), propylene glycol monolaurate (PGML), N-methyl 2-pyrrolidone (NMP), Miglyol 812N® (MG), and mineral oil (MO). Single, binary or ternary systems were examined. The same vehicles were subsequently examined to investigate niacinamide delivery in vivo. For this proof-of-concept study one donor was used for the in vitro studies and one volunteer for the in vivo investigations to minimise biovariability. Analysis of in vitro samples was conducted using HPLC and in vivo uptake of niacinamide was evaluated using Confocal Raman spectroscopy (CRS). The amount of niacinamide permeated through skin in vitro was linearly proportional to the intensity of the niacinamide signal determined in the stratum corneum in vivo. A good correlation was observed between the signal intensities of selected vehicles and niacinamide signal intensity. The findings provide further support for the use of CRS to monitor drug delivery into and across the skin. In addition, the results highlight the critical role of the vehicle and its disposition in skin for effective dermal delivery.

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

    Science.gov (United States)

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

    2015-11-10

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

  16. Electromagnetized gold nanoparticles mediate direct lineage reprogramming into induced dopamine neurons in vivo for Parkinson's disease therapy

    Science.gov (United States)

    Yoo, Junsang; Lee, Euiyeon; Kim, Hee Young; Youn, Dong-Ho; Jung, Junghyun; Kim, Hongwon; Chang, Yujung; Lee, Wonwoong; Shin, Jaein; Baek, Soonbong; Jang, Wonhee; Jun, Won; Kim, Soochan; Hong, Jongki; Park, Hi-Joon; Lengner, Christopher J.; Moh, Sang Hyun; Kwon, Youngeun; Kim, Jongpil

    2017-10-01

    Electromagnetic fields (EMF) are physical energy fields generated by electrically charged objects, and specific ranges of EMF can influence numerous biological processes, which include the control of cell fate and plasticity. In this study, we show that electromagnetized gold nanoparticles (AuNPs) in the presence of specific EMF conditions facilitate an efficient direct lineage reprogramming to induced dopamine neurons in vitro and in vivo. Remarkably, electromagnetic stimulation leads to a specific activation of the histone acetyltransferase Brd2, which results in histone H3K27 acetylation and a robust activation of neuron-specific genes. In vivo dopaminergic neuron reprogramming by EMF stimulation of AuNPs efficiently and non-invasively alleviated symptoms in mouse Parkinson's disease models. This study provides a proof of principle for EMF-based in vivo lineage conversion as a potentially viable and safe therapeutic strategy for the treatment of neurodegenerative disorders.

  17. Fluorescence In Vivo Hybridization (FIVH) for Detection of Helicobacter pylori Infection in a C57BL/6 Mouse Model

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Leite, Marina; Cappoen, Davie

    2016-01-01

    ). Finally, the efficiency of FIVH to detect H. pylori SS1 strain in C57BL/6 infected mice was evaluated ex vivo in mucus samples, in cryosections and paraffin-embedded sections by epifluorescence and confocal microscopy. RESULTS: H. pylori SS1 strain infecting C57BL/6 mice was successfully detected...... by the Cy3_HP_LNA/2OMe_PS probe in the mucus, attached to gastric epithelial cells and colonizing the gastric pits. The specificity of the probe for H. pylori was confirmed by microscopy. CONCLUSIONS: In the future this methodology can be used in combination with a confocal laser endomicroscope for in vivo......INTRODUCTION: In this study, we applied fluorescence in vivo hybridization (FIVH) using locked nucleic acid (LNA) probes targeting the bacterial rRNA gene for in vivo detection of H. pylori infecting the C57BL/6 mouse model. A previously designed Cy3_HP_LNA/2OMe_PS probe, complementary...

  18. DNA stress and strain, in silico, in vitro and in vivo

    International Nuclear Information System (INIS)

    Levens, David; Benham, Craig J

    2011-01-01

    A vast literature has explored the genetic interactions among the cellular components regulating gene expression in many organisms. Early on, in the absence of any biochemical definition, regulatory modules were conceived using the strict formalism of genetics to designate the modifiers of phenotype as either cis- or trans-acting depending on whether the relevant genes were embedded in the same or separate DNA molecules. This formalism distilled gene regulation down to its essence in much the same way that consideration of an ideal gas reveals essential thermodynamic and kinetic principles. Yet just as the anomalous behavior of materials may thwart an engineer who ignores their non-ideal properties, schemes to control and manipulate the genetic and epigenetic programs of cells may falter without a fuller and more quantitative elucidation of the physical and chemical characteristics of DNA and chromatin in vivo

  19. Modeling the in vivo case with in vitro nanotoxicity data.

    Science.gov (United States)

    Shelley, Michael L; Wagner, Andrew J; Hussain, Saber M; Bleckmann, Charles

    2008-01-01

    As more in vitro nanotoxicity data appear in the literature, these findings must be translated to in vivo effects to define nanoparticle exposure risk. Physiologically based pharmacokinetic (PBPK) modeling has played a significant role in guiding and validating in vivo studies for molecular chemical exposure and can develop as a significant tool in guiding similar nanotoxicity studies. This study models the population dynamics of a single cell type within a specific tissue. It is the first attempt to model the in vitro effects of a nanoparticle exposure, in this case aluminum (80 nm) and its impact on a population of rat alveolar macrophages (Wagner et al. 2007, J. Phys. Chem. B 111:7353-7359). The model demonstrates how in vitro data can be used within a simulation setting of in vivo cell dynamics and suggests that PBPK models should be developed quickly to interpret nanotoxicity data, guide in vivo study design, and accelerate nanoparticle risk assessment.

  20. Preparation, Characterisation and In Vivo Evaluation of Silybin ...

    African Journals Online (AJOL)

    HP

    The nanoparticles sustained the release of the drug both in vitro and in vivo for up to 10 days and offered better ... without affecting normal ECM synthesis, effectively delivered ... oral bioavailability problems with sylibin can be avoided since ...

  1. An evaluation of preference for video and in vivo modeling.

    Science.gov (United States)

    Geiger, Kaneen B; Leblanc, Linda A; Dillon, Courtney M; Bates, Stephanie L

    2010-01-01

    We assessed preference for video or in vivo modeling using a concurrent-chains arrangement with 3 children with autism. The two modeling conditions produced similar acquisition rates and no differential selection (i.e., preference) for all 3 participants.

  2. Cystoscopic optical coherence tomography for urinary bladder imaging in vivo

    Science.gov (United States)

    Wang, Z. G.; Adler, H.; Chan, D.; Jain, A.; Xie, H. K.; Wu, Z. L.; Pan, Y. T.

    2006-02-01

    This paper summarizes the development of new 2D MEMS mirrors and the pertinent modification to improve OCT endoscopic catheter packaging suitable for in vivo imaging diagnosis of bladder cancers. Comparative study of the newly developed endocopic OCT versus the bench-top OCT is presented. Results of in vivo OCT cystoscopy based on a porcine acute inflammation model are presented to compare time-domain OCT and spectral-domain OCT for in vivo imaging. In addition, results of spectral-domain Doppler OCT are presented to image blood flow in the lamina propria of the bladder. The results of our in vivo animal study using the presented OCT endoscope are discussed for potential problems in the future clinical applications.

  3. In vivo antiplasmodial effect of chloroform extracts of Artemisia ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-01

    Dec 1, 2009 ... Full Length Research Paper. In vivo antiplasmodial effect of ... responsible for over a million deaths each year (Breman,. 2001). In tropical and ... A major breakthrough of the past decades has been the discovery by Chinese ...

  4. High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.

    Directory of Open Access Journals (Sweden)

    Michelle Cronin

    Full Text Available The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT for interpretation.In this study, the non-pathogenic commensal bacteria E. coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (i.v. administered to mice bearing subcutaneous (s.c FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post i.v.-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.

  5. In vitro and in vivo antioxidant activities of inulin

    OpenAIRE

    Shang, Hong-Mei; Zhou, Hai-Zhu; Yang, Jun-Yan; Li, Ran; Song, Hui; Wu, Hong-Xin

    2018-01-01

    This study was designed to investigate the in vitro and in vivo antioxidant activities of inulin. The in vitro assays demonstrated that the antioxidant activities of inulin, including the DPPH radical scavenging activity, ABTS scavenging activity and ferric reducing power, were weak and significantly lower than those of Vitamin C (P < 0.05). The influence of dietary supplementation with inulin on the antioxidant status of laying hens was evaluated with in vivo antioxidant assays. The results ...

  6. Oncology In Vivo Data Integration for Hypothesis Generation

    Directory of Open Access Journals (Sweden)

    Wei Jia

    2012-06-01

    Full Text Available AstraZeneca’s Oncology in vivo data integration platform brings multidimensional data from animal model efficacy, pharmacokinetic and pharmacodynamic data to animal model profiling data and public in vivo studies. Using this platform, scientists can cluster model efficacy and model profiling data together, quickly identify responder profiles and correlate molecular characteristics to pharmacological response. Through meta-analysis, scientists can compare pharmacology between single and combination treatments, between different drug scheduling and administration routes.