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Sample records for bronchoepithelium egfp expression

  1. BAC-Dkk3-EGFP Transgenic Mouse: An In Vivo Analytical Tool for Dkk3 Expression

    Directory of Open Access Journals (Sweden)

    Yuki Muranishi

    2012-01-01

    Full Text Available Dickkopf (DKK family proteins are secreted modulators of the Wnt signaling pathway and are capable of regulating the development of many organs and tissues. We previously identified Dkk3 to be a molecule predominantly expressed in the mouse embryonic retina. However, which cell expresses Dkk3 in the developing and mature mouse retina remains to be elucidated. To examine the precise expression of the Dkk3 protein, we generated BAC-Dkk3-EGFP transgenic mice that express EGFP integrated into the Dkk3 gene in a BAC plasmid. Expression analysis using the BAC-Dkk3-EGFP transgenic mice revealed that Dkk3 is expressed in retinal progenitor cells (RPCs at embryonic stages and in Müller glial cells in the adult retina. Since Müller glial cells may play a potential role in retinal regeneration, BAC-Dkk3-EGFP mice could be useful for retinal regeneration studies.

  2. Direct visualization of membrane architecture of myelinating cells in transgenic mice expressing membrane-anchored EGFP.

    Science.gov (United States)

    Deng, Yaqi; Kim, BongWoo; He, Xuelian; Kim, Sunja; Lu, Changqing; Wang, Haibo; Cho, Ssang-Goo; Hou, Yiping; Li, Jianrong; Zhao, Xianghui; Lu, Q Richard

    2014-04-01

    Myelinogenesis is a complex process that involves substantial and dynamic changes in plasma membrane architecture and myelin interaction with axons. Highly ramified processes of oligodendrocytes in the central nervous system (CNS) make axonal contact and then extrapolate to wrap around axons and form multilayer compact myelin sheathes. Currently, the mechanisms governing myelin sheath assembly and axon selection by myelinating cells are not fully understood. Here, we generated a transgenic mouse line expressing the membrane-anchored green fluorescent protein (mEGFP) in myelinating cells, which allow live imaging of details of myelinogenesis and cellular behaviors in the nervous systems. mEGFP expression is driven by the promoter of 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNP) that is expressed in the myelinating cell lineage. Robust mEGFP signals appear in the membrane processes of oligodendrocytes in the CNS and Schwann cells in the peripheral nervous system (PNS), wherein mEGFP expression defines the inner layers of myelin sheaths and Schmidt-Lanterman incisures in adult sciatic nerves. In addition, mEGFP expression can be used to track the extent of remyelination after demyelinating injury in a toxin-induced demyelination animal model. Taken together, the membrane-anchored mEGFP expression in the new transgenic line would facilitate direct visualization of dynamic myelin membrane formation and assembly during development and process remodeling during remyelination after various demyelinating injuries.

  3. 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...

  4. Potential utility of eGFP-expressing NOG mice (NOG-EGFP as a high purity cancer sampling system

    Directory of Open Access Journals (Sweden)

    Shima Kentaro

    2012-06-01

    Full Text Available Abstract Purpose It is still technically difficult to collect high purity cancer cells from tumor tissues, which contain noncancerous cells. We hypothesized that xenograft models of NOG mice expressing enhanced green fluorescent protein (eGFP, referred to as NOG-EGFP mice, may be useful for obtaining such high purity cancer cells for detailed molecular and cellular analyses. Methods Pancreato-biliary cancer cell lines were implanted subcutaneously to compare the tumorigenicity between NOG-EGFP mice and nonobese diabetic/severe combined immunodeficiency (NOD/SCID mice. To obtain high purity cancer cells, the subcutaneous tumors were harvested from the mice and enzymatically dissociated into single-cell suspensions. Then, the cells were sorted by fluorescence-activated cell sorting (FACS for separation of the host cells and the cancer cells. Thereafter, the contamination rate of host cells in collected cancer cells was quantified by using FACS analysis. The viability of cancer cells after FACS sorting was evaluated by cell culture and subsequent subcutaneous reimplantation in NOG-EGFP mice. Results The tumorigenicity of NOG-EGFP mice was significantly better than that of NOD/SCID mice in all of the analyzed cell lines (p  Conclusions This method provides a novel cancer sampling system for molecular and cellular analysis with high accuracy and should contribute to the development of personalized medicine.

  5. Compact whole-body fluorescent imaging of nude mice bearing EGFP expressing tumor

    Science.gov (United States)

    Chen, Yanping; Xiong, Tao; Chu, Jun; Yu, Li; Zeng, Shaoqun; Luo, Qingming

    2005-01-01

    Issue of tumor has been a hotspot of current medicine. It is important for tumor research to detect tumors bearing in animal models easily, fast, repetitively and noninvasivly. Many researchers have paid their increasing interests on the detecting. Some contrast agents, such as green fluorescent protein (GFP) and Discosoma red fluorescent protein (Dsred) were applied to enhance image quality. Three main kinds of imaging scheme were adopted to visualize fluorescent protein expressing tumors in vivo. These schemes based on fluorescence stereo microscope, cooled charge-coupled-device (CCD) or camera as imaging set, and laser or mercury lamp as excitation light source. Fluorescence stereo microscope, laser and cooled CCD are expensive to many institutes. The authors set up an inexpensive compact whole-body fluorescent imaging tool, which consisted of a Kodak digital camera (model DC290), fluorescence filters(B and G2;HB Optical, Shenyang, Liaoning, P.R. China) and a mercury 50-W lamp power supply (U-LH50HG;Olympus Optical, Japan) as excitation light source. The EGFP was excited directly by mercury lamp with D455/70 nm band-pass filter and fluorescence was recorded by digital camera with 520nm long-pass filter. By this easy operation tool, the authors imaged, in real time, fluorescent tumors growing in live mice. The imaging system is external and noninvasive. For half a year our experiments suggested the imaging scheme was feasible. Whole-body fluorescence optical imaging for fluorescent expressing tumors in nude mouse is an ideal tool for antitumor, antimetastatic, and antiangiogenesis drug screening.

  6. Establishment of a hepatocellular carcinoma cell line expressing dual reporter genes: sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP)

    International Nuclear Information System (INIS)

    Kwak, Won Jung; Koo, Bon Chul; Kwon, Mo Sun

    2007-01-01

    Dual reporter gene imaging has several advantages for more sophisticated molecular imaging studies such as gene therapy monitoring. Herein, we have constructed hepatoma cell line expressing dual reporter genes of sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP), and the functionalities of the genes were evaluated in vivo by nuclear and optical imaging. A pRetro-PN vector was constructed after separating NIS gene from pcDNA-NIS. RSV-EGFP-WPRE fragment separated from pLNRGW was cloned into pRetro-PN vector. The final vector expressing dual reporter genes was named pRetro-PNRGW. A human hepatoma (HepG2) cells were transfected by the retrovirus containing NIS and EGFP gene (HepG2-NE). Expression of NIS gene was confirmed by RT-PCR, radioiodine uptake and efflux studies. Expression of EGFP was confirmed by RT-PCR and fluorescence microscope. The HepG2 and HepG2-NE cells were implanted in shoulder and hindlimb of nude mice, then fluorescence image, gamma camera image and I-124 microPET image were undertaken. The HepG2-NE cell was successfully constructed. RT-PCR showed NIS and EGFP mRNA expression. About 50% of cells showed fluorescence. The iodine uptake of NIS-expressed cells was about 9 times higher than control. In efflux study, T 1/2 of HepG2-NE cells was 9 min. HepG2-NE xenograft showed high signal-to-background fluorescent spots and higher iodine-uptake compared to those of HepG2 xenograft. A hepatoma cell line expressing NIS and EGFP dual reporter genes was successfully constructed and could be used as a potential either by therapeutic gene or imaging reporter gene

  7. pLIVE-EGFP: A liver specific vector carrying the EGFP reporter for ...

    African Journals Online (AJOL)

    -EGFP gene at the Xho I site of the pLIVE vector. The pLIVE-EGFP vector permits simultaneous expression of a gene of interest in addition to the EGFP reporter, specifically within liver cells, both in vivo and in vitro. When expressed in liver cells ...

  8. eGFP expression under the Uchl1 promoter labels corticospinal motor neurons and a subpopulation of degeneration resistant spinal motor neurons in ALS mouse models

    Science.gov (United States)

    Yasvoina, Marina V.

    Current understanding of basic cellular and molecular mechanisms for motor neuron vulnerability during motor neuron disease initiation and progression is incomplete. The complex cytoarchitecture and cellular heterogeneity of the cortex and spinal cord greatly impedes our ability to visualize, isolate, and study specific neuron populations in both healthy and diseased states. We generated a novel reporter line, the Uchl1-eGFP mouse, in which cortical and spinal components of motor neuron circuitry are genetically labeled with eGFP under the Uchl1 promoter. A series of cellular and anatomical analyses combined with retrograde labeling, molecular marker expression, and electrophysiology were employed to determine identity of eGFP expressing cells in the motor cortex and the spinal cord of novel Uchl1-eGFP reporter mice. We conclude that eGFP is expressed in corticospinal motor neurons (CSMN) in the motor cortex and a subset of S-type alpha and gamma spinal motor neurons (SMN) in the spinal cord. hSOD1G93A and Alsin-/- mice, mouse models for amyotrophic lateral sclerosis (ALS), were bred to Uchl1-eGFP reporter mouse line to investigate the pathophysiology and underlying mechanisms of CSMN degeneration in vivo. Evidence suggests early and progressive degeneration of CSMN and SMN in the hSOD1G93A transgenic mice. We show an early increase of autophagosome formation in the apical dendrites of vulnerable CSMN in hSOD1G93A-UeGFP mice, which is localized to the apical dendrites. In addition, labeling S-type alpha and gamma SMN in the hSOD1G93A-UeGFP mice provide a unique opportunity to study basis of their resistance to degeneration. Mice lacking alsin show moderate clinical phenotype and mild CSMN axon degeneration in the spinal cord, which suggests vulnerability of CSMN. Therefore, we investigated the CSMN cellular and axon defects in aged Alsin-/- mice bred to Uchl1-eGFP reporter mouse line. We show that while CSMN are preserved and lack signs of degeneration, CSMN axons

  9. Morphological Analysis of the Axonal Projections of EGFP-Labeled Esr1-Expressing Neurons in Transgenic Female Medaka.

    Science.gov (United States)

    Zempo, Buntaro; Karigo, Tomomi; Kanda, Shinji; Akazome, Yasuhisa; Oka, Yoshitaka

    2018-02-01

    Some hypothalamic neurons expressing estrogen receptor α (Esr1) are thought to transmit a gonadal estrogen feedback signal to gonadotropin-releasing hormone 1 (GnRH1) neurons, which is the final common pathway for feedback regulation of reproductive functions. Moreover, estrogen-sensitive neurons are suggested to control sexual behaviors in coordination with reproduction. In mammals, hypothalamic estrogen-sensitive neurons release the peptide kisspeptin and regulate GnRH1 neurons. However, a growing body of evidence in nonmammalian species casts doubt on the regulation of GnRH1 neurons by kisspeptin neurons. As a step toward understanding how estrogen regulates neuronal circuits for reproduction and sex behavior in vertebrates in general, we generated a transgenic (Tg) medaka that expresses enhanced green fluorescent protein (EGFP) specifically in esr1-expressing neurons (esr1 neurons) and analyzed their axonal projections. We found that esr1 neurons in the preoptic area (POA) project to the gnrh1 neurons. We also demonstrated by transcriptome and histological analyses that these esr1 neurons are glutamatergic or γ-aminobutyric acidergic (GABAergic) but not kisspeptinergic. We therefore suggest that glutamatergic and GABAergic esr1 neurons in the POA regulate gnrh1 neurons. This hypothesis is consistent with previous studies in mice that found that glutamatergic and GABAergic transmission is critical for estrogen-dependent changes in GnRH1 neuron firing. Thus, we propose that this neuronal circuit may provide an evolutionarily conserved mechanism for regulation of reproduction. In addition, we showed that telencephalic esr1 neurons project to medulla, which may control sexual behavior. Moreover, we found that some POA-esr1 neurons coexpress progesterone receptors. These neurons may form the neuronal circuits that regulate reproduction and sex behavior in response to the serum estrogen/progesterone. Copyright © 2018 Endocrine Society.

  10. Core labeling of adenovirus with EGFP

    International Nuclear Information System (INIS)

    Le, Long P.; Le, Helen N.; Nelson, Amy R.; Matthews, David A.; Yamamoto, Masato; Curiel, David T.

    2006-01-01

    The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expression vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology

  11. Distinct expression/function of potassium and chloride channels contributes to the diverse volume regulation in cortical astrocytes of GFAP/EGFP mice

    Czech Academy of Sciences Publication Activity Database

    Benešová, Jana; Rusňáková, Vendula; Honsa, Pavel; Pivoňková, Helena; Džamba, Dávid; Kubista, Mikael; Anděrová, Miroslava

    2012-01-01

    Roč. 7, č. 1 (2012), e29725 E-ISSN 1932-6203 R&D Projects: GA ČR GAP303/10/1338 Grant - others:GA ČR(CZ) GD309/08/H079 Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50520701 Institutional support: RVO:86652036 ; RVO:68378041 Keywords : cell volume regulation * GFAP/EGFP * ischemia Subject RIV: FH - Neurology; FH - Neurology (BTO-N) Impact factor: 3.730, year: 2012

  12. Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone.

    Science.gov (United States)

    Harel-Markowitz, Eliane; Gurevich, Michael; Shore, Laurence S; Katz, Adi; Stram, Yehuda; Shemesh, Mordechai

    2009-05-01

    Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

  13. Establishment and characterization of CAG/EGFP transgenic rabbit line.

    Science.gov (United States)

    Takahashi, Ri-ichi; Kuramochi, Takashi; Aoyagi, Kazuki; Hashimoto, Shu; Miyoshi, Ichiro; Kasai, Noriyuki; Hakamata, Yoji; Kobayashi, Eiji; Ueda, Masatsugu

    2007-02-01

    Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP fluorescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.

  14. The dark side of EGFP: defective polyubiquitination.

    Directory of Open Access Journals (Sweden)

    Mathijs Baens

    Full Text Available Enhanced Green Fluorescent Protein (EGFP is the most commonly used live cell reporter despite a number of conflicting reports that it can affect cell physiology. Thus far, the precise mechanism of GFP-associated defects remained unclear. Here we demonstrate that EGFP and EGFP fusion proteins inhibit polyubiquitination, a posttranslational modification that controls a wide variety of cellular processes, like activation of kinase signalling or protein degradation by the proteasome. As a consequence, the NF-kappaB and JNK signalling pathways are less responsive to activation, and the stability of the p53 tumour suppressor is enhanced in cell lines and in vivo. In view of the emerging role of polyubiquitination in the regulation of numerous cellular processes, the use of EGFP as a live cell reporter should be carefully considered.

  15. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    International Nuclear Information System (INIS)

    Watanabe, Masahito; Umeyama, Kazuhiro; Matsunari, Hitomi; Takayanagi, Shuko; Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka; Nakauchi, Hiromitsu

    2010-01-01

    Research highlights: → EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. → ZFNs induced targeted mutations in porcine primary cultured cells. → Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  16. Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiaoxia [Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); University of Chinese Academy of Sciences, Beijing (China); Chen, Xiaowen; Jin, Xia; He, Jiangyan [Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Yin, Zhan, E-mail: zyin@ihb.ac.cn [Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Ningbo Laboratory, State Key Laboratory of Freshwater Ecology and Biotechnology (China)

    2014-07-01

    The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were all identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO{sub 4}, dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. - Highlights: • Identification of the promoter of zebrafish glycoprotein subunit α (gsuα) gene • Generation of stable transmission gsuα:EGFP transgenic zebrafish reporter • Demonstration of the recapitulation of the gsuα:EGFP and endogenous gsuα expression • Suggestion of the gsuα:EGFP transgenic zebrafish as a novel platform for EDC study.

  17. Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

    International Nuclear Information System (INIS)

    Cheng, Xiaoxia; Chen, Xiaowen; Jin, Xia; He, Jiangyan; Yin, Zhan

    2014-01-01

    The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were all identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO 4 , dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. - Highlights: • Identification of the promoter of zebrafish glycoprotein subunit α (gsuα) gene • Generation of stable transmission gsuα:EGFP transgenic zebrafish reporter • Demonstration of the recapitulation of the gsuα:EGFP and endogenous gsuα expression • Suggestion of the gsuα:EGFP transgenic zebrafish as a novel platform for EDC study

  18. Transformation of Beauveria bassiana to produce EGFP in Tenebrio molitor for use as animal feed additives.

    Science.gov (United States)

    Kim, Jae Su; Choi, Jae Young; Lee, Se Jin; Lee, Ju Hyun; Fu, Zhenli; Skinner, Margaret; Parker, Bruce L; Je, Yeon Ho

    2013-07-01

    Efforts are underway to develop more effective and safer animal feed additives. Entomopathogenic fungi can be considered practical expression platforms of functional genes within insects which have been used as animal feed additives. In this work, as a model, the enhanced green fluorescent protein (egfp) gene was expressed in yellow mealworms, Tenebrio molitor by highly infective Beauveria bassiana ERL1170. Among seven test isolates, ERL1170 treatment showed 57.1% and 98.3% mortality of mealworms 2 and 5 days after infection, respectively. The fungal transformation vector, pABeG containing the egfp gene, was inserted into the genomic DNA of ERL1170 using the restriction enzyme-mediated integration method. This resulted in the generation of the transformant, Bb-egfp#3, which showed the highest level of fluorescence. Bb-egfp#3-treated mealworms gradually turned dark brown, and in 7-days mealworm sections showed a strong fluorescence. This did not occur in the wild-type strain. This work suggests that further valuable proteins can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the animal feed additive industry. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  19. Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

    Science.gov (United States)

    Ganini, Douglas; Leinisch, Fabian; Kumar, Ashutosh; Jiang, JinJie; Tokar, Erik J; Malone, Christine C; Petrovich, Robert M; Mason, Ronald P

    2017-08-01

    Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H 2 O 2 ) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H 2 O 2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O 2 •- ) and H 2 O 2 in the presence of NADH. Generation of the free radical O 2 •- and H 2 O 2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies. Published by Elsevier B.V.

  20. Establishment of a rabbit Oct4 promoter-based EGFP reporter system.

    Directory of Open Access Journals (Sweden)

    Longquan Quan

    Full Text Available Rabbits are commonly used as laboratory animal models to investigate human diseases and phylogenetic development. However, pluripotent stem cells that contribute to germline transmission have yet to be established in rabbits. The transcription factor Oct4, also known as Pou5f1, is considered essential for the maintenance of the pluripotency of stem cells. Hence, pluripotent cells can be identified by monitoring Oct4 expression using a well-established Oct4 promoter-based reporter system. This study developed a rabbit Oct4 promoter-based enhanced green fluorescent protein (EGFP reporter system by transfecting pROP2-EGFP into rabbit fetal fibroblasts (RFFs. The transgenic RFFs were used as donor cells for somatic cell nuclear transfer (SCNT. The EGFP expression was detected in the blastocysts and genital ridges of SCNT fetuses. Fibroblasts and neural stem cells (NSCs were derived from the SCNT fetuses. EGFP was also reactivated in blastocysts after the second SCNT, and induced pluripotent stem cells (iPSCs were obtained after reprogramming using Yamanaka's factors. The results above indicated that a rabbit reporter system used to monitor the differentiating status of cells was successfully developed.

  1. Trichomonas vaginalis virus-mediated expression of EGFP in the parasitic protozoan Trichomonas vagi-nails%阴道毛滴虫病毒介导的EGFP在阴道毛滴虫细胞内的表达

    Institute of Scientific and Technical Information of China (English)

    郭瑞娟; 李淑红; 张西臣; 李建华; 宫鹏涛; 杨举; 张国才

    2010-01-01

    目的 研究阴道毛滴虫病毒(Trichomonas vaginalis virus,TVV)介导外源基因进入阴道毛滴虫体内表达的能力,探索TVV作为双链RNA病毒转染载体的可能性.方法 根据TVV基因组的序列特征,用绿色荧光蛋白(EG-FP)编码基因替换TVV的全部或部分基因编码区,构建TVV与增强型EGFP编码基因的嵌合体pTVV-EGFP,其体外转录体经电穿孔方法转染携病毒阴道毛滴虫株,RT-PCR及SDS-PAGE方法检测EGFP的表达情况.结果 电穿孔转染后培养的虫体在荧光显微镜下观察到绿色荧光信号,且续传15代后仍然存在;RT-PCR检测到EGFP的mRNA,SDS-PAGE检测到转染后虫体及培养上清中有分子质量单位为27 ku的蛋白,与已知EGFP的分子质量相符.结论 TVV能成功介导外源性EGFP编码基因在阴道毛滴虫体内表达.

  2. Use of TSHβ:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Cheng [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Graduate University of Chinese Academy of Sciences, Beijing (China); Jin, Xia; He, Jiangyan [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Yin, Zhan, E-mail: zyin@ihb.ac.cn [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China)

    2012-07-15

    Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit β (TSHβ) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSHβ in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSHβ promoter, the similar responsive patterns between the expression levels of TSHβ:EGFP and endogenous TSHβ mRNA in the pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSHβ:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system. Highlights: ► The promoter of zebrafish TSHβ gene has been identified. ► The stable TSHβ:EGFP transgenic zebrafish reporter germline has been generated. ► The EGFP in the transgenic fish recapitulated the pattern of pituitary TSHβ mRNA. ► The transgenic zebrafish may be an in vivo model for EDC assessment.

  3. Use of TSHβ:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals

    International Nuclear Information System (INIS)

    Ji, Cheng; Jin, Xia; He, Jiangyan; Yin, Zhan

    2012-01-01

    Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit β (TSHβ) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSHβ in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSHβ promoter, the similar responsive patterns between the expression levels of TSHβ:EGFP and endogenous TSHβ mRNA in the pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSHβ:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system. Highlights: ► The promoter of zebrafish TSHβ gene has been identified. ► The stable TSHβ:EGFP transgenic zebrafish reporter germline has been generated. ► The EGFP in the transgenic fish recapitulated the pattern of pituitary TSHβ mRNA. ► The transgenic zebrafish may be an in vivo model for EDC assessment.

  4. The sustained-release behavior and in vitro and in vivo transfection of pEGFP-loaded core-shell-structured chitosan-based composite particles

    Science.gov (United States)

    Wang, Yun; Lin, Fu-xing; Zhao, Yu; Wang, Mo-zhen; Ge, Xue-wu; Gong, Zheng-xing; Bao, Dan-dan; Gu, Yu-fang

    2014-01-01

    Novel submicron core-shell-structured chitosan-based composite particles encapsulated with enhanced green fluorescent protein plasmids (pEGFP) were prepared by complex coacervation method. The core was pEGFP-loaded thiolated N-alkylated chitosan (TACS) and the shell was pH- and temperature-responsive hydroxybutyl chitosan (HBC). pEGFP-loaded TACS-HBC composite particles were spherical, and had a mean diameter of approximately 120 nm, as measured by transmission electron microscopy and particle size analyzer. pEGFP showed sustained release in vitro for >15 days. Furthermore, in vitro transfection in human embryonic kidney 293T and human cervix epithelial cells, and in vivo transfection in mice skeletal muscle of loaded pEGFP, were investigated. Results showed that the expression of loaded pEGFP, both in vitro and in vivo, was slow but could be sustained over a long period. pEGFP expression in mice skeletal muscle was sustained for >60 days. This work indicates that these submicron core-shell-structured chitosan-based composite particles could potentially be used as a gene vector for in vivo controlled gene transfection. PMID:25364253

  5. Generation of Col2a1-EGFP iPS cells for monitoring chondrogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Taku Saito

    Full Text Available Induced pluripotent stem cells (iPSC are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP transgenic mice by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. Among the 30 clones of Col2a1-EGFP iPSC we established, two clones showed high expression levels of embryonic stem cell (ESC marker genes, similar to control ESC. A teratoma formation assay showed that the two clones were pluripotent and differentiated into cell types from all three germ layers. The fluorescent signal was observed during chondrogenic differentiation of the two clones concomitant with the increase in chondrocyte marker expression. In conclusion, Col2a1-EGFP iPSC are useful for monitoring chondrogenic differentiation and will contribute to research in cartilage regenerative medicine.

  6. The sustained-release behavior and in vitro and in vivo transfection of pEGFP-loaded core-shell-structured chitosan-based composite particles

    Directory of Open Access Journals (Sweden)

    Wang Y

    2014-10-01

    Full Text Available Yun Wang,1 Fu-xing Lin,2 Yu Zhao,1 Mo-zhen Wang,2 Xue-wu Ge,2 Zheng-xing Gong,1 Dan-dan Bao,1 Yu-fang Gu1 1Department of Plastic Surgery, First Affiliated Hospital of Anhui Medical University, 2CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui, People’s Republic of China Abstract: Novel submicron core-shell-structured chitosan-based composite particles ­encapsulated with enhanced green fluorescent protein plasmids (pEGFP were prepared by complex coacervation method. The core was pEGFP-loaded thiolated N-alkylated chitosan (TACS and the shell was pH- and temperature-responsive hydroxybutyl chitosan (HBC. pEGFP-loaded TACS-HBC composite particles were spherical, and had a mean diameter of approximately 120 nm, as measured by transmission electron microscopy and particle size analyzer. pEGFP showed sustained release in vitro for >15 days. Furthermore, in vitro transfection in human embryonic kidney 293T and human cervix epithelial cells, and in vivo transfection in mice skeletal muscle of loaded pEGFP, were investigated. Results showed that the expression of loaded pEGFP, both in vitro and in vivo, was slow but could be sustained over a long period. pEGFP expression in mice skeletal muscle was sustained for >60 days. This work indicates that these submicron core-shell-structured chitosan-based composite particles could potentially be used as a gene vector for in vivo controlled gene transfection. Keywords: gene therapy, gene transfection, hydroxybutyl chitosan, thiolated N-alkylated chitosan, pEGFP, complex coacervation

  7. Polyethylene glycol (PEG)-mediated transformation of the fused egfp ...

    African Journals Online (AJOL)

    Yomi

    2012-03-06

    Mar 6, 2012 ... pUC19 vector. Enhanced green fluorescent protein-encoding gene (egfp) and a selection marker, the hygromycin ... agro-industrial waste bioconversion (Shabtay et al., 2009;. Salvachúa et al., 2011); and .... gene, two pairs of primers were designed based on the Lentinula edodes sequences published by ...

  8. An Evaluation on Transfection Efficiency of pHRE-Egr1-EGFP in Hepatocellular Carcinoma Cells Bel-7402 Mediated by PEI-MZF-NPs

    Directory of Open Access Journals (Sweden)

    Mei Lin

    2011-01-01

    Full Text Available To improve transfection and expression efficiency of target gene, especially under cancer anoxic microenvironment, we have developed pHRE-Egr1-EGFP/PEI-MZF-NPs nanosystem, in which pHRE-Egr1-EGFP, eukaryotic gene expression plasmid, is constructed by combining radiation promoter Egr1 with anoxia induction components (HRE, forming anoxic radiation double sensitive HRE/Egr1 promoter to activate reporter gene EGFP expression. MZF-NPs (Mn0.5 Zn0.5 Fe2O4 magnetic nanoparticles, obtained by coprecipitation method, are coated with cation poly(ethylenimine (PEI. We transferred pHRE-Egr1-EGFP into hepatocellular carcinoma Bel-7402 cells, using PEI-MZF-NPs as the carrier and tested some relevant efficacy. The results show that PEI-MZF-NPs have good DNA-binding ability, protection ability, release ability, little toxicity, and high transfection efficiency, obviously superior to those of the liposome method and electricity perforation method. Moreover, the expression level of EGFP gene induced by anoxia and radiation was significantly higher than that of single radiation activation. It is therefore concluded that HRE/Egr1 can induce and improve target gene expression efficiency in cancer anoxic microenvironment, and that PEI-MZF-NPs can be used as a novel nonviral gene vector which offers a viable approach to the mediated radiation gene therapy of cancer.

  9. Differential tissue expression of enhanced green fluorescent protein in ‘Green mice’

    OpenAIRE

    Ma, De-Fu; Tezuka, Hideo; Kondo, Tetsuo; Sudo, Katsuko; Niu, Dong-Feng; Nakazawa, Tadao; Kawasaki, Tomonori; Yamane, Tetsu; Nakamura, Nobuki; Katoh, Ryohei

    2010-01-01

    In order to clarify tissue expression of enhanced green fluorescent protein (EGFP) in ‘green mice’ from a transgenic line having an EGFP cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer, we studied the expression of EGFP in various organs and tissues from these ‘green mice’ by immunohistochemistry with anti- EGFP antibody in conjunction with direct observation for EGFP fluorescence using confocal laser scanning microscopy. On i...

  10. Adipogenic differentiation and EGFP gene transfection of amniotic fluid-derived stem cells from goat fetus at terminal gestational age.

    Science.gov (United States)

    He, Xiao-Ying; Zheng, Yue-Mao; Qiu, Shuang; Qi, Ying-Pei; Zhang, Yong

    2011-08-01

    The aims of this study were to determine whether stem cells could be isolated from amniotic fluid of goat fetus at terminal gestational age and to determine if these stem cells could differentiate into adipogenic cells and be transfected with a reporter gene, EGFP (enhanced green fluorescent protein). The stem cells were isolated from amniotic fluid of goat fetus at terminal gestational age, induced to differentiate into adipogenic cells in vitro and transfected with the EGFP gene using lipofection. Markers associated with undifferentiated AFS (amniotic fluid-derived stem) cells were tested by RT (reverse transcription)-PCR. The results demonstrated that AFS cells could be isolated from amniotic fluid of goat fetus at terminal gestational age and could differentiate into adipogenic cells. The EGFP gene was transfected into AFS cells successfully. EGFP gene transfection efficiency of the three groups of transgenic AFS cells were 26.0, 29.9 and 30.5%, respectively. Both transgenic and wild-type AFS cells could express Hes1 (hairy and enhancer of split 1), Oct4 (octamer-binding protein 4) and Nanog.

  11. pLIVE-EGFP: A liver specific vector carrying the EGFP reporter for ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... system for monitoring gene expression, intracellular protein trafficking or localization. Several types of reporter systems, such as secretory alkaline phosphatase (SEAP), luciferase, β-galactosidase, or fluorescent proteins (FPs), have been widely applied in these researches (Naylor,. 1999; Branchini, et al., ...

  12. SPONTANEOUS AND MNNG-INDUCED REVERSION OF AN EGFP CONSTRUCT IN HELA CELLS: AN ASSAY FOR OBSERVING MUTATIONS IN LIVING CELLS BY FLUORESCENT MICROSCOPY

    Science.gov (United States)

    A HeLa cell line stably expressing the Enhanced Green Fluorescence Protein (EGFP) gene, interrupted by the IVS2-654 intron, was studied without treatment and after treatment with a single standard dose of 15 ?M of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done ...

  13. Feasibility of dual reporter gene in rat myoblast cell line using human sodium iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) gene

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yong Jin; Lee, You La; Ahn, Sohn Joo; Choi, Chang Ik; Lee, Sang Woo; Ahn, Byeong Cheol; Lee, Jae Tae [School of Medicine, Kyungpook National University, Daegu (Korea, Republic of)

    2007-07-01

    To develop a non-invasive combined imaging method of gamma camera and optical imaging to assess rat myoblast cell line, H9c2, we constructed retrovirus containing hNIS and EGFP gene, and transfected to rat myoblast cell and monitored hNIS and EGFP expression. Rat myoblast cell line, H9C2, was transfected with hNIS and EGFP gene using retrovirus (H9C2-NG). The expression of hNIS and EGFP gene was determined by RT-PCR and fluorescence microscopy, respectively. The uptake and efflux of I-125 were measured in the transfected and wild type cell lines. Each cell line was injected to 4 flank sites (H9c2: 1X107 or 2X107, H9C2-NG: 1X107 or 2X107) in nude mouse. Scintigraphic image was performed at 3h, 1 day after H9C2 and H9C2-NG cell inoculation. We performed gamma camera and animal PET imaging to evaluate NIS expression. Also, GFP image obtained using optical imaging system. The expression of hNIS and EGFP gene was confirmed by RT-PCR. In iodide uptake, H9C2-NG cells accumulated 274.52.2 pmol/ mg protein at 30 min. But wild type cell line did not uptake iodide. In fluorescent microscopy, H9C2-NG cells were highly fluorescent than that of H9C2 cells. In iodide efflux study, 50% of radioactivity flowed out during the first 10min. Scintigraphy showed increased uptake of Tc-99m in H9c2-NG than in H9C2 for 1 day. Also, H9C2-NG cells showed high signal-to-background fluorescent spots in animal body. In this study, NIS and EGFP reporter gene were successfully transfected by a retrovirus in myoblast cell line, and the transfected cell can be easily visualized in vivo. These results suggest that NIS and EGFP gene has an excellent feasibility as a reporter gene, and it can be used to monitor cell trafficking for monitoring.

  14. Development of the 5-HT2CR-Tango System Combined with an EGFP Reporter Gene.

    Science.gov (United States)

    Watanabe, Yoshihisa; Tsujimura, Atsushi; Aoki, Miku; Taguchi, Katsutoshi; Tanaka, Masaki

    2016-02-01

    The serotonin 2C receptor (5-HT2CR) is a G-protein-coupled receptor implicated in emotion, feeding, reward, and cognition. 5-HT2CRs are pharmacological targets for mental disorders and metabolic and reward system abnormalities, as alterations in 5-HT2CR expression, RNA editing, and SNPs are involved in these disturbances. To date, 5-HT2CR activity has mainly been measured by quantifying inositol phosphate production and intracellular Ca(2+) release, but these assays are not suitable for in vivo analysis. Here, we developed a 5-HT2CR-Tango assay system, a novel analysis tool of 5-HT2CR activity based on the G-protein-coupled receptor (GPCR)-arrestin interaction. With desensitization of activated 5-HT2CR by arrestin, this system converts the 5-HT2CR-arrestin interaction into EGFP reporter gene signal via the LexA transcriptional activation system. For validation of our system, we measured activity of two 5-HT2CR RNA-editing isoforms (INI and VGV) in HEK293 cells transfected with EGFP reporter gene. The INI isoform displayed both higher basal- and 5-HT-stimulated activities than the VGV isoform. Moreover, an inhibitory effect of 5-HT2CR antagonist SB242084 was also detected by 5-HT2CR-Tango system. This novel tool is useful for in vitro high-throughput targeted 5-HT2CR drug screening and can be applied to future in vivo brain function studies associated with 5-HT2CRs in transgenic animal models.

  15. Fluorescence-Activated Cell Sorting of EGFP-Labeled Neural Crest Cells From Murine Embryonic Craniofacial Tissue

    Directory of Open Access Journals (Sweden)

    Saurabh Singh

    2005-01-01

    Full Text Available During the early stages of embryogenesis, pluripotent neural crest cells (NCC are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. Crossing Wnt1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt1-Cre transgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR. The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development.

  16. Generation of Pax6-IRES-EGFP knock-in mouse via the cloning-free CRISPR/Cas9 system to reliably visualize neurodevelopmental dynamics.

    Science.gov (United States)

    Inoue, Yukiko U; Morimoto, Yuki; Hoshino, Mikio; Inoue, Takayoshi

    2018-07-01

    Pax6 encodes a transcription factor that plays pivotal roles in eye development, early brain patterning, neocortical arealization, and so forth. Visualization of Pax6 expression dynamics in these events could offer numerous advantages to neurodevelopmental studies. While CRISPR/Cas9 system has dramatically accelerated one-step generation of knock-out mouse, establishment of gene-cassette knock-in mouse via zygote injection has been considered insufficient due to its low efficiency. Recently, an improved CRISPR/Cas9 system for effective gene-cassette knock-in has been reported, where the native form of guide RNAs (crRNA and tracrRNA) assembled with recombinant Cas9 protein are directly delivered into mouse fertilized eggs. Here we apply this strategy to insert IRES-EGFP-pA cassette into Pax6 locus and achieve efficient targeted insertions of the 1.8 kb reporter gene. In Pax6-IRES-EGFP mouse we have generated, EGFP-positive cells reside in the eyes and cerebellum as endogenous Pax6 expressing cells at postnatal day 2. At the early embryonic stages when the embryos are transparent, EGFP-positive regions can be easily identified without PCR-based genotyping, precisely recapitulating the endogenous Pax6 expression patterns. Remarkably, at E12.5, the graded expression patterns of Pax6 in the developing neocortex now become recognizable in our knock-in mice, serving a sufficiently sensitive and useful tool to precisely visualize neurodevelopmental processes. Copyright © 2018 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.

  17. Enhanced green fluorescent protein is a nearly ideal long-term expression tracer for hematopoietic stem cells, whereas DsRed-express fluorescent protein is not.

    Science.gov (United States)

    Tao, Wen; Evans, Barbara-Graham; Yao, Jing; Cooper, Scott; Cornetta, Kenneth; Ballas, Christopher B; Hangoc, Giao; Broxmeyer, Hal E

    2007-03-01

    Validated gene transfer and expression tracers are essential for elucidating functions of mammalian genes. Here, we have determined the suitability and unintended side effects of enhanced green fluorescent protein (EGFP) and DsRed-Express fluorescent protein as expression tracers in long-term hematopoietic stem cells (HSCs). Retrovirally transduced mouse bone marrow cells expressing either EGFP or DsRed-Express in single or mixed dual-color cell populations were clearly discerned by flow cytometry and fluorescence microscopy. The results from in vivo competitive repopulation assays demonstrated that EGFP-expressing HSCs were maintained nearly throughout the lifespan of the transplanted mice and retained long-term multilineage repopulating potential. All mice assessed at 15 months post-transplantation were EGFP positive, and, on average, 24% total peripheral white blood cells expressed EGFP. Most EGFP-expressing recipient mice lived at least 22 months. In contrast, Discosoma sp. red fluorescent protein (DsRed)-expressing donor cells dramatically declined in transplant-recipient mice over time, particularly in the competitive setting, in which mixed EGFP- and DsRed-expressing cells were cotransplanted. Moreover, under in vitro culture condition favoring preservation of HSCs, purified EGFP-expressing cells grew robustly, whereas DsRed-expressing cells did not. Therefore, EGFP has no detectable deteriorative effects on HSCs, and is nearly an ideal long-term expression tracer for hematopoietic cells; however, DsRed-Express fluorescent protein is not suitable for these cells.

  18. Altered astrocytic swelling in the cortex of α-syntrophin-negative GFAP/EGFP mice.

    Directory of Open Access Journals (Sweden)

    Miroslava Anderova

    Full Text Available Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K(+ and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4. As the removal of perivascular AQP4 via the deletion of α-syntrophin was shown to delay edema formation and K(+ clearance, we aimed to elucidate the impact of α-syntrophin knockout on volume changes in individual astrocytes in situ evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α in situ and in vivo in the mouse cortex employing the real-time iontophoretic method. RT-qPCR profiling was used to reveal possible differences in the expression of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. To visualize individual astrocytes in mice lacking α-syntrophin we crossbred GFAP/EGFP mice, in which the astrocytes are labeled by the enhanced green fluorescent protein under the human glial fibrillary acidic protein promoter, with α-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that α-syntrophin deletion results in significantly smaller astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD or 50 mM K(+. As for the mild stimuli, such as mild hypoosmotic or hyperosmotic stress or 10 mM K(+, α-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative α changes showed a significantly smaller decrease in α-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of α-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K(+.

  19. Differential tissue expression of enhanced green fluorescent protein in 'green mice'.

    Science.gov (United States)

    Ma, De-Fu; Tezuka, Hideo; Kondo, Tetsuo; Sudo, Katsuko; Niu, Dong-Feng; Nakazawa, Tadao; Kawasaki, Tomonori; Yamane, Tetsu; Nakamura, Nobuki; Katoh, Ryohei

    2010-06-01

    In order to clarify tissue expression of enhanced green fluorescent protein (EGFP) in 'green mice' from a transgenic line having an EGFP cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer, we studied the expression of EGFP in various organs and tissues from these 'green mice' by immunohistochemistry with anti- EGFP antibody in conjunction with direct observation for EGFP fluorescence using confocal laser scanning microscopy. On immunohistochemical examination and on direct observation by confocal laser scanning microscopy, the level of EGFP expression varied among organs and tissues. EGFP expression was diffusely and strongly observed in the skin, pituitary, thyroid gland, parathyroid gland, heart, gall bladder, pancreas, adrenals and urinary bladder. There was only sporadic and weak expression of EGFP in the epithelium of the trachea, bronchus of the lung, stratified squamous epithelium and gastric glands of the stomach, hepatic bile ducts of the liver, glomeruli and renal tubules of the kidney and endo-metrial glands of the uterus. Furthermore, EGFP was only demonstrated within the goblet and paneth cells in the colon and small intestine, the tall columnar cells in the ductus epididymis, and the leydig cells in the testis. In conclusion, our results show that EGFP is differentially expressed in organs and tissues of 'green mice', which indicates that 'green mice' may prove useful for research involving transplantation and tissue clonality.

  20. Impact of pEGFP mediated ING4 gene on growth of glioma U251 ...

    African Journals Online (AJOL)

    Jane

    2011-06-22

    Jun 22, 2011 ... Full Length Research Paper. Impact of pEGFP mediated ING4 gene on growth of glioma U251 cells and its potential molecular mechanism. Yuefei Deng*, Bingxi Lei and Yiying Zhao. Department of Neurosurgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China.

  1. Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

    Directory of Open Access Journals (Sweden)

    Kawakami Minoru

    2011-11-01

    Full Text Available Abstract Background Neural crest cells (NCCs are embryonic, multipotent stem cells. Their long-range and precision-guided migration is one of their most striking characteristics. We previously reported that P0-Cre/CAG-CAT-lacZ double-transgenic mice showed significant lacZ expression in tissues derived from NCCs. Results In this study, by embedding a P0-Cre/CAG-CAT-EGFP embryo at E9.5 in collagen gel inside a culture glass slide, we were able to keep the embryo developing ex vivo for more than 24 hours; this development was with enough NCC fluorescent signal intensity to enable single-cell resolution analysis, with the accompanying NCC migration potential intact and with the appropriate NCC response to the extracellular signal maintained. By implantation of beads with absorbed platelet-derived growth factor-AA (PDGF-AA, we demonstrated that PDGF-AA acts as an NCC-attractant in embryos. We also performed assays with NCCs isolated from P0-Cre/CAG-CAT-EGFP embryos on culture plates. The neuromediator 5-hydroxytryptamine (5-HT has been known to regulate NCC migration. We newly demonstrated that dopamine, in addition to 5-HT, stimulated NCC migration in vitro. Two NCC populations, with different axial levels of origins, showed unique distribution patterns regarding migration velocity and different dose-response patterns to both 5-HT and dopamine. Conclusions Although avian species predominated over the other species in the NCC study, our novel system should enable us to use mice to assay many different aspects of NCCs in embryos or on culture plates, such as migration, division, differentiation, and apoptosis.

  2. Transgenic fluorescent zebrafish Tg(fli1:EGFP)y¹ for the identification of vasotoxicity within the zFET.

    Science.gov (United States)

    Delov, Vera; Muth-Köhne, Elke; Schäfers, Christoph; Fenske, Martina

    2014-05-01

    The fish embryo toxicity test (FET) is currently one of the most advocated animal alternative tests in ecotoxicology. To date, the application of the FET with zebrafish (zFET) has focused on acute toxicity assessment, where only lethal morphological effects are accounted for. An application of the zFET beyond acute toxicity, however, necessitates the establishment of more refined and quantifiable toxicological endpoints. A valuable tool in this context is the use of gene expression-dependent fluorescent markers that can even be measured in vivo. We investigated the application of embryos of Tg(fli1:EGFP)(y1) for the identification of vasotoxic substances within the zFET. Tg(fli1:EGFP)(y1) fish express enhanced GFP in the entire vasculature under the control of the fli1 promoter, and thus enable the visualization of vascular defects in live zebrafish embryos. We assessed the fli1 driven EGFP-expression in the intersegmental blood vessels (ISVs) qualitatively and quantitatively, and found an exposure concentration related increase in vascular damage for chemicals like triclosan, cartap and genistein. The fluorescence endpoint ISV-length allowed an earlier and more sensitive detection of vasotoxins than the bright field assessment method. In combination with the standard bright field morphological effect assessment, an increase in significance and value of the zFET for a mechanism-specific toxicity evaluation was achieved. This study highlights the benefits of using transgenic zebrafish as convenient tools for identifying toxicity in vivo and to increase sensitivity and specificity of the zFET. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

    Science.gov (United States)

    Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

    2016-02-01

    Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time

  4. Time-resolved stimulated emission depletion and energy transfer dynamics in two-photon excited EGFP

    Science.gov (United States)

    Masters, T. A.; Robinson, N. A.; Marsh, R. J.; Blacker, T. S.; Armoogum, D. A.; Larijani, B.; Bain, A. J.

    2018-04-01

    Time and polarization-resolved stimulated emission depletion (STED) measurements are used to investigate excited state evolution following the two-photon excitation of enhanced green fluorescent protein (EGFP). We employ a new approach for the accurate STED measurement of the hitherto unmeasured degree of hexadecapolar transition dipole moment alignment ⟨α40 ⟩ present at a given excitation-depletion (pump-dump) pulse separation. Time-resolved polarized fluorescence measurements as a function of pump-dump delay reveal the time evolution of ⟨α40 ⟩ to be considerably more rapid than predicted for isotropic rotational diffusion in EGFP. Additional depolarization by homo-Förster resonance energy transfer is investigated for both ⟨α20 ⟩ (quadrupolar) and ⟨α40 ⟩ transition dipole alignments. These results point to the utility of higher order dipole correlation measurements in the investigation of resonance energy transfer processes.

  5. Adipose-derived stem cells transfected with pEGFP-OSX enhance bone formation during distraction osteogenesis*

    OpenAIRE

    Lai, Qing-guo; Sun, Shao-long; Zhou, Xiao-hong; Zhang, Chen-ping; Yuan, Kui-feng; Yang, Zhong-jun; Luo, Sheng-lei; Tang, Xiao-peng; Ci, Jiang-bo

    2014-01-01

    This study was designed to investigate the effects of local delivery of adipose-derived stem cells (ADSCs) transfected with transcription factor osterix (OSX) on bone formation during distraction osteogenesis. New Zealand white rabbits (n=54) were randomly divided into three groups (18 rabbits per group). A directed cloning technique was used for the construction of recombinant plasmid pEGFP-OSX, where EGFP is the enhanced green fluorescence protein. After osteodistraction of the right mandib...

  6. A transgenic approach to study argininosuccinate synthetase gene expression

    Science.gov (United States)

    2014-01-01

    Background Argininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed. Results Two lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage

  7. Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord.

    Science.gov (United States)

    Ronsyn, Mark W; Daans, Jasmijn; Spaepen, Gie; Chatterjee, Shyama; Vermeulen, Katrien; D'Haese, Patrick; Van Tendeloo, Viggo Fi; Van Marck, Eric; Ysebaert, Dirk; Berneman, Zwi N; Jorens, Philippe G; Ponsaerts, Peter

    2007-12-14

    Bone marrow-derived stromal cells (MSC) are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord. First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSC-NT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants. In this study, we demonstrate that genetically modified hMSC lines can survive

  8. Expression and purification of recombinant nattokinase in Spodoptera frugiperda cells.

    Science.gov (United States)

    Li, Xiaoxiang; Wang, Xiaoli; Xiong, Shaoling; Zhang, Jing; Cai, Litao; Yang, Yanyan

    2007-10-01

    A recombinant baculovirus, rv-egfp-NK, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP), was used to express nattokinase (NK), a fibrinolytic enzyme, in Spodoptera frugiperda (SF-9) cells. The recombinant protein also included a histidine tag for purification using Ni(2+) resins. The recombinant NK, approximately 30 kDa, retained fibrinolytic activity (60 U/ml). The integration of the EGFP expression cassette in the Bac-to-Bac system is thus an effective method for the expression and purification of recombinant NK protein in Spodoptera frugiperda insect cells.

  9. Visualization of the endocytic pathway in the filamentous fungus Aspergillus oryzae using an EGFP-fused plasma membrane protein

    International Nuclear Information System (INIS)

    Higuchi, Yujiro; Nakahama, Tomoyuki; Shoji, Jun-ya; Arioka, Manabu; Kitamoto, Katsuhiko

    2006-01-01

    Endocytosis is an important process for cellular activities. However, in filamentous fungi, the existence of endocytosis has been so far elusive. In this study, we used AoUapC-EGFP, the fusion protein of a putative uric acid-xanthine permease with enhanced green fluorescent protein (EGFP) in Aspergillus oryzae, to examine whether the endocytic process occurs or not. Upon the addition of ammonium into the medium the fusion protein was internalized from the plasma membrane. The internalization of AoUapC-EGFP was completely blocked by sodium azide, cold, and cytochalasin A treatments, suggesting that the internalization possesses the general features of endocytosis. These results demonstrate the occurrence of endocytosis in filamentous fungi. Moreover, we discovered that the endosomal compartments appeared upon the induction of endocytosis and moved in a microtubule-dependent manner

  10. Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

    Directory of Open Access Journals (Sweden)

    Vuento Matti

    2006-12-01

    Full Text Available Abstract Fluorescence correlation spectroscopy (FCS monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids were fused to the C-terminus of the enhanced green fluorescent protein (EGFP. The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs. The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

  11. Chimeric analysis of EGFP and DsRed2 transgenic mice demonstrates polyclonal maintenance of pancreatic acini.

    Science.gov (United States)

    Ryu, Je-Young; Siswanto, Antoni; Harimoto, Kenichi; Tagawa, Yoh-ichi

    2013-06-01

    The pancreatic islet is an assembly of specific endocrine cells. There are many conflicting reports regarding whether the acinus develops from single or multiple progenitor cells. This study investigated the development and maintenance clonality of the pancreatic acinus and duct using a chimeric analysis with EGFP and DsRed2 transgenic mice. Chimeric mice (G-R mice) were obtained by the aggregation method, using 8-cell stage embryos from EGFP and DsRed2 transgenic mice. The islets from the G-R mice were chimeric and mosaic, consisting of either EGFP- or DsRed2-positive populations, as in previous reports. On the other hand, most acini developed from either EGFP or DsRed2 origin, but some were chimeric. Interestingly, these chimeric acini were clearly separated into two-color regions and were not mosaic. Some large intralobular pancreatic ducts consisting of more than 10 cells were found to be chimeric, but no small ducts made up of less than 9 cells were chimeric. Our histological observations suggest that the pancreatic acinus polyclonally and directionally is maintained by multiple progenitor cells. Pancreatic large ducts also seem to develop polyclonally and might result from the assembly of small ducts that develop from a single origin. These findings provide useful information for further understanding pancreatic maintenance.

  12. Enhanced EGFP Fluorescence Emission in Presence of PEG Aqueous Solutions and PIB1000-PEG6000-PIB1000 Copolymer Vesicles

    Directory of Open Access Journals (Sweden)

    Noor Muhammad

    2013-01-01

    Full Text Available An EGFP construct interacting with the PIB1000-PEG6000-PIB1000 vesicles surface reported a ~2-fold fluorescence emission enhancement. Because of the constructs nature with the amphiphilic peptide inserted into the PIB core, EGFP is expected to experience a “pure” PEG environment. To unravel this phenomenon PEG/water solutions at different molecular weights and concentrations were used. Already at ~1 : 10 protein/PEG molar ratio the increase in fluorescence emission is observed reaching a plateau correlating with the PEG molecular weight. Parallel experiments in presence of glycerol aqueous solutions did show a slight fluorescence enhancement however starting at much higher concentrations. Molecular dynamics simulations of EGFP in neat water, glycerol, and PEG aqueous solutions were performed showing that PEG molecules tend to “wrap” the protein creating a microenvironment where the local PEG concentration is higher compared to its bulk concentration. Because the fluorescent emission can be perturbed by the refractive index surrounding the protein, the clustering of PEG molecules induces an enhanced fluorescence emission already at extremely low concentrations. These findings can be important when related to the use of EGFP as reported in molecular biology experiments.

  13. Transfection of HeLa-cells with pEGFP plasmid by impedance power-assisted electroporation

    DEFF Research Database (Denmark)

    Glahder, Jacob; Norrild, Bodil; Persson, Mikael B

    2005-01-01

    Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 microg DNA in 90 microl low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and puls...

  14. Gene expression profiles of Bapx1 expressing FACS sorted cells from wildtype and Bapx1-EGFP null mouse embryos

    Directory of Open Access Journals (Sweden)

    Sumantra Chatterjee

    2015-09-01

    Full Text Available The data described in this article refers to Chatterjee et al. (2015 “In vivo genome-wide analysis of multiple tissues identifies gene regulatory networks, novel functions and downstream regulatory genes for Bapx1 and its co-regulation with Sox9 in the mammalian vertebral column” (GEO GSE35649 [1]. Transcriptional profiling combined with genome wide binding data is a powerful tool to elucidate the molecular mechanism behind vertebrate organogenesis. It also helps to uncover multiple roles of a single gene in different organs. In the above mentioned report we reveal the function of the homeobox gene Bapx1 during the embryogenesis of five distinct organs (vertebral column, spleen, gut, forelimb and hindlimb at a relevant developmental stage (E12.5, microarray analysis of isolated wildtype and mutant cells in is compared in conjunction with ChIP-Seq analysis. We also analyzed the development of the vertebral column by comparing microarray and ChIP-Seq data for Bapx1 with similarly generated data sets for Sox9 to generate a gene regulatory network controlling various facets of the organogenesis.

  15. Experimental study on transferring pEGFP into mice facial nerve by ultrasound-mediated microbubbles%超声微泡对pEGFP质粒转染小鼠损伤面神经的促进作用

    Institute of Scientific and Technical Information of China (English)

    王丹; 骆文龙

    2011-01-01

    目的:探讨超声微泡介导基因转染面神经的可行性及有效性。方法:建立小鼠面神经损伤模型,以增强型绿色荧光蛋白质粒(pEGFP)为标记基因。将48只小鼠随机分为6组:空白组(A组)、脂质体+质粒组(B组)、微泡+质粒+超声组(C组)、微泡+质粒组(D组)、超声+质粒组(E组)、单纯质粒组(F组)。将小鼠面神经损伤后,局部注射吸附pEGFP质粒的微泡,行超声辐照,3天后,取出面神经干行快速冰冻切片,激光共聚焦显微镜下观察各组绿色荧光蛋白的表达,并行平均荧光强度分析,并采用western blot法检测面神经干绿色荧光蛋白的表达。同时在激光共聚焦显微镜下观察各组小鼠脑干绿色荧光蛋白表达。结果:微泡+质粒+超声组面神经干平均荧光强度最强,其次分别为脂质体+质粒组、超声+质粒组、微泡+质粒组、单纯质粒组、空白组。各组间两两比较均有统计学意义(P〈0.05或0.01)。脑干尚未发现面神经核团有明显绿色荧光蛋白表达。结论:微泡在超声的作用下能够安全、有效地将目的基因转染进入损伤后的面神经。%Objective:To investigate the feasibility and efficacy of transferring pEGFP into mice facial nerve by ultrasound-mediated microbubbles.Methods! Exposure,clamp bilateral facial nerve trunks to perform the injury of the mice' s facial nerve.Plasmid enhanced green fluorescent protein(pEGFP) was chose to reflect the efficacy of transfection.48 mice were randomly divided into six groups: surgery only group(A),lipofectamineTM 2000+pEGFP group(B),Ultrasound+ Microbubbles+pEGFPgroup(C),Microbubbles+pEGFPgroup(D),Ultrasound+pEGFPgroup(E) and pEGFP only treatment group(F).The bilateral facial nerve trunks were harvested at 3rd day after treatment respectively.The expression of pEGFP in facial nerve trunks was examined by laser

  16. [Expression of mutation type GJA8 gene and wild type GJA8 gene of a congenital inherited nuclear cataract family in eukaryotic cells].

    Science.gov (United States)

    Zheng, Jian-qiu; Liu, Ping; Wang, Jian-wen; Liu, Jian-ju

    2010-04-20

    To clone the sequence of mutation type GJA8 gene (mGJA8) and wild type GJA8 gene (wGJA8) of a congenital inherited nuclear cataract family and study their expression in eukaryotic cell lines in vitro. The mGJA8 and wGJA8 were amplified from this family's DNA and healthy people's DNA by PCR respectively. The mGJA8 and wGJA8 were recombined with plasmid pEGFP-N1 respectively. The accuracy of pEGFP-N1-GJA8 was confirmed by restriction enzyme digestion and DNA sequencing. Finally pEGFP-N1- mGJA8 and pEGFP-N1- wGJA8 and GFP protein were transfected into COS7 cells by lipofectin. The expression of pEGFP-N1-GJA8 and GFP fusion protein were to observe under fluorescence microscope, and to detect by Western-blotting and immunohistochemical staining. The mGJA8 and wGJA8 were cloned successfully. With restricting enzyme digestion analysis and DNA sequencing, recombinant plasmid pEGFP-N1-mGJA8 and pEGFP-N1-wGJA8 were constructed correctly and their GFP fusions were expressed in transfected COS7 cells. The expression of pEGFP-N1-mGJA8 and pEGFP-N1-wGJA8 fusion protein were observed under fluorescence microscope, and detected by Western-blotting and immunohistochemical staining successfully. The mGJA8 gene and wGJA8 gene are cloned successfully, and pEGFP-N1-mGJA8 and pEGFP-N1-mGJA8 fusion protein can be expressed in COS7 cells, which establish the foundation for further studying the mechanism of this congenital inherited nuclear cataract family.

  17. TALEN/CRISPR-mediated eGFP knock-in add-on at the OCT4 locus does not impact differentiation of human embryonic stem cells towards endoderm.

    Directory of Open Access Journals (Sweden)

    Nicole A J Krentz

    Full Text Available Human embryonic stem cells (hESCs have great promise as a source of unlimited transplantable cells for regenerative medicine. However, current progress on producing the desired cell type for disease treatment has been limited due to an insufficient understanding of the developmental processes that govern their differentiation, as well as a paucity of tools to systematically study differentiation in the lab. In order to overcome these limitations, cell-type reporter hESC lines will be required. Here we outline two strategies using Transcription Activator Like Effector Nucleases (TALENs and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-CRISPR-Associated protein (Cas to create OCT4-eGFP knock-in add-on hESC lines. Thirty-one and forty-seven percent of clones were correctly modified using the TALEN and CRISPR-Cas9 systems, respectively. Further analysis of three correctly targeted clones demonstrated that the insertion of eGFP in-frame with OCT4 neither significantly impacted expression from the wild type allele nor did the fusion protein have a dramatically different biological stability. Importantly, the OCT4-eGFP fusion was easily detected using microscopy, flow cytometry and western blotting. The OCT4 reporter lines remained equally competent at producing CXCR4+ definitive endoderm that expressed a panel of endodermal genes. Moreover, the genomic modification did not impact the formation of NKX6.1+/SOX9+ pancreatic progenitor cells following directed differentiation. In conclusion, these findings demonstrate for the first time that CRISPR-Cas9 can be used to modify OCT4 and highlight the feasibility of creating cell-type specific reporter hESC lines utilizing genome-editing tools that facilitate homologous recombination.

  18. Chemical reactivation of resin-embedded pHuji adds red for simultaneous two-color imaging with EGFP

    Science.gov (United States)

    Guo, Wenyan; Liu, Xiuli; Liu, Yurong; Gang, Yadong; He, Xiaobin; Jia, Yao; Yin, Fangfang; Li, Pei; Huang, Fei; Zhou, Hongfu; Wang, Xiaojun; Gong, Hui; Luo, Qingming; Xu, Fuqiang; Zeng, Shaoqun

    2017-01-01

    The pH-sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EGFP or EYFP is good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is an urgent need. Here a pH-sensitive red fluorescent protein, pHuji, is selected and verified to remain pH-sensitive in HM20 resin. We observe 183% fluorescence intensity of pHuji in resin-embeded mouse brain and 29.08-fold fluorescence intensity of reactivated pHuji compared to the quenched state. pHuji and EGFP can be quenched and chemically reactivated simultaneously in resin, thus enabling simultaneous two-color micro-optical sectioning tomography of resin-embedded mouse brain. This method may greatly facilitate the visualization of neuronal morphology and neural circuits to promote understanding of the structure and function of the brain. PMID:28717566

  19. Dynamic expression of Lgr6 in the developing and mature mouse cochlea

    Science.gov (United States)

    Zhang, Yanping; Chen, Yan; Ni, Wenli; Guo, Luo; Lu, Xiaoling; Liu, Liman; Li, Wen; Sun, Shan; Wang, Lei; Li, Huawei

    2015-01-01

    The Wnt/β-catenin signaling pathway plays important roles in mammalian inner ear development. Lgr5, one of the downstream target genes of the Wnt/β-catenin signaling pathway, has been reported to be a marker for inner ear hair cell progenitors. Lgr6 shares approximately 50% sequence homology with Lgr5 and has been identified as a stem cell marker in several organs. However, the detailed expression profiles of Lgr6 have not yet been investigated in the mouse inner ear. Here, we first used Lgr6-EGFP-Ires-CreERT2 mice to examine the spatiotemporal expression of Lgr6 protein in the cochlear duct during embryonic and postnatal development. Lgr6-EGFP was first observed in one row of prosensory cells in the middle and basal turn at embryonic day 15.5 (E15.5). From E18.5 to postnatal day 3 (P3), the expression of Lgr6-EGFP was restricted to the inner pillar cells (IPCs). From P7 to P15, the Lgr6-EGFP expression level gradually decreased in the IPCs and gradually increased in the inner border cells (IBCs). At P20, Lgr6-EGFP was only expressed in the IBCs, and by P30 Lgr6-EGFP expression had completely disappeared. Next, we demonstrated that Wnt/β-catenin signaling is required to maintain the Lgr6-EGFP expression in vitro. Finally, we demonstrated that the Lgr6-EGFP-positive cells isolated by flow cytometry could differentiate into myosin 7a-positive hair cells after 10 days in-culture, and this suggests that the Lgr6-positive cells might serve as the hair cell progenitor cells in the cochlea. PMID:26029045

  20. Dynamic Expression of Lgr6 in the Developing and Mature Mouse Cochlea

    Directory of Open Access Journals (Sweden)

    Yanping eZhang

    2015-05-01

    Full Text Available The Wnt/β-catenin signaling pathway plays important roles in mammalian inner ear development. Lgr5, one of the downstream target genes of the Wnt/β-catenin signaling pathway, has been reported to be a marker for inner ear hair cell progenitors. Lgr6 shares approximately 50% sequence homology with Lgr5 and has been identified as a stem cell marker in several organs. However, the detailed expression profiles of Lgr6 have not yet been investigated in the mouse inner ear. Here, we first used Lgr6-EGFP-Ires-CreERT2 mice to examine the spatiotemporal expression of Lgr6 protein in the cochlear duct during embryonic and postnatal development. Lgr6-EGFP was first observed in one row of prosensory cells in the middle and basal turn at embryonic day 15.5 (E15.5. From E18.5 to postnatal day 3 (P3, the expression of Lgr6-EGFP was restricted to the inner pillar cells (IPCs. From P7 to P15, the Lgr6-EGFP expression level gradually decreased in the IPCs and gradually increased in the inner border cells (IBCs. At P20, Lgr6-EGFP was only expressed in the IBCs, and by P30 Lgr6-EGFP expression had completely disappeared. Next, we demonstrated that Wnt/β-catenin signaling is required to maintain the Lgr6-EGFP expression in vitro. Finally, we demonstrated that the Lgr6-EGFP-positive cells isolated by flow cytometry could differentiate into myosin 7a-positive hair cells after 10 days in-culture, and this suggests that the Lgr6-positive cells might serve as the hair cell progenitor cells in the cochlea.

  1. Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue.

    Science.gov (United States)

    Yee, Karen K; Li, Yan; Redding, Kevin M; Iwatsuki, Ken; Margolskee, Robert F; Jiang, Peihua

    2013-05-01

    Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5-expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate (CV) and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5-expressing cells in posterior tongue are a subset of K14-positive epithelial cells. Lineage-tracing experiments using an inducible Cre knockin allele in combination with Rosa26-LacZ and Rosa26-tdTomato reporter strains showed that Lgr5-expressing cells gave rise to taste cells, perigemmal cells, along with self-renewing cells at the bottom of trench areas at the base of CV and foliate papillae. Moreover, using subtype-specific taste markers, we found that Lgr5-expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue. Copyright © 2013 AlphaMed Press.

  2. Over-expression of brain-derived neurotrophic factor in mesenchymal stem cells transfected with recombinant lentivirus BDNF gene.

    Science.gov (United States)

    Zhang, X; Zhu, J; Zhang, K; Liu, T; Zhang, Z

    2016-12-30

    This study was aimed at investigating the expression of brain-derived neurotrophic factor (BDNF) in mesenchymal stem cells (MSCs) modified with recombinant lentivirus bearing BDNF gene. Lentivirus vectors bearing BDNF gene were constructed. MSCs were isolated from rats and cultured. The lentiviral vectors containing BDNF gene were transfected into the MSCs, and BDNF gene and protein expressions were monitored with enhanced green fluorescent protein (EGFP). RT-PCR and Western blot were used to measure gene and protein expressions, respectibvely in MSCs, MSCs-EGFP and MSCs-EGFP-BDNF groups. Green fluorescence assay confirmed successful transfection of BDNF gene recombinant lentivirus into MSCs. RT-PCR and Western blot revealed that BDNF gene and protein expressions in the MSCs-EGFP-BDNF group were significantly higher than that in MSCs group and MSCs-EGFP group. There were no statistically significant differences in gene expression between MSCs and MSCs-EGFP groups. MSCs can over-express BDNF when transfected with recombinant lentivirus bearing BDNF gene.

  3. Production and characterization of active recombinant interleukin-12/eGFP fusion protein in stably-transfected DF1 chicken cells.

    Science.gov (United States)

    Wu, Hsing Chieh; Chen, Yu San; Shen, Pin Chun; Shien, Jui Hung; Lee, Long Huw; Chiu, Hua Hsien

    2015-01-01

    The adjuvant activity of chicken interleukin-12 (chIL-12) protein has been described as similar to that of mammalian IL-12. Recombinant chIL-12 can be produced using several methods, but chIL-12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL-12 which stably expressed a fusion protein, chIL-12 and enhanced green fluorescent protein (eGFP) connected by a (G4 S)3 linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10(6) DF1/chIL-12 cells were inoculated in a T-175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL(-1) and 2,207 ± 3.28 ng mL(-1) , respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN-γ, which was measured using an enzyme-linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL-12 cells with DMSO or producing chIL-12 in a fusion protein form does not have adverse effects on the bioactivity of chIL-12. © 2015 American Institute of Chemical Engineers.

  4. Functional characterisation of filamentous actin probe expression in neuronal cells.

    Directory of Open Access Journals (Sweden)

    Shrujna Patel

    Full Text Available Genetically encoded filamentous actin probes, Lifeact, Utrophin and F-tractin, are used as tools to label the actin cytoskeleton. Recent evidence in several different cell types indicates that these probes can cause changes in filamentous actin dynamics, altering cell morphology and function. Although these probes are commonly used to visualise actin dynamics in neurons, their effects on axonal and dendritic morphology has not been systematically characterised. In this study, we quantitatively analysed the effect of Lifeact, Utrophin and F-tractin on neuronal morphogenesis in primary hippocampal neurons. Our data show that the expression of actin-tracking probes significantly impacts on axonal and dendrite growth these neurons. Lifeact-GFP expression, under the control of a pBABE promoter, caused a significant decrease in total axon length, while another Lifeact-GFP expression, under the control of a CAG promoter, decreased the length and complexity of dendritic trees. Utr261-EGFP resulted in increased dendritic branching but Utr230-EGFP only accumulated in cell soma, without labelling any neurites. Lifeact-7-mEGFP and F-tractin-EGFP in a pEGFP-C1 vector, under the control of a CMV promoter, caused only minor changes in neuronal morphology as detected by Sholl analysis. The results of this study demonstrate the effects that filamentous actin tracking probes can have on the axonal and dendritic compartments of neuronal cells and emphasise the care that must be taken when interpreting data from experiments using these probes.

  5. Generation of H1 PAX6WT/EGFP reporter cells to purify PAX6 positive neural stem/progenitor cells.

    Science.gov (United States)

    Wu, Wei; Liu, Juli; Su, Zhenghui; Li, Zhonghao; Ma, Ning; Huang, Ke; Zhou, Tiancheng; Wang, Linli

    2018-08-25

    Neural conversion from human pluripotent cells (hPSCs) is a potential therapy to neurological disease in the future. However, this is still limited by efficiency and stability of existed protocols used for neural induction from hPSCs. To overcome this obstacle, we developed a reporter system to screen PAX6 + neural progenitor/stem cells using transcription activator like effector nuclease (TALEN). We found that knock-in 2 A-EGFP cassette into PAX6 exon of human embryonic stem cells H1 with TALEN-based homology recombination could establish PAX6 WT/EGFP H1 reporter cell line fast and efficiently. This reporter cell line could differentiate into PAX6 and EGFP double positive neural progenitor/stem cells (NPCs/NSCs) after neural induction. Those PAX6 WT/EGFP NPCs could be purified, expanded and specified to post-mitotic neurons in vitro efficiently. With this reporter cell line, we also screened out 1 NPC-specific microRNA, hsa-miR-99a-5p, and 3 ESCs-enriched miRNAs, hsa-miR-302c-5p, hsa-miR-512-3p and hsa-miR-518 b. In conclusion, the TALEN-based neural stem cell screening system is safe and efficient and could help researcher to acquire adequate and pure neural progenitor cells for further application. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. (AAV)-mediated expression of small interfering RNA

    African Journals Online (AJOL)

    Effective inhibition of specific gene by adenoassociated virus (AAV)-mediated expression of small interfering RNA. ... To perform functional tests on siRNA, which was expressed by the viral vector, recombinant AAVs, coding for siRNA against exogenous gene, EGFP, and endogenous gene, p53, were established and ...

  7. Synthesis, characterization, and cytotoxicity of the plasmid EGFP-p53 loaded on pullulan–spermine magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Eslaminejad, Touba, E-mail: tslaminejad@yahoo.com [Pharmaceutics Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Nematollahi-Mahani, Seyed Noureddin, E-mail: nnematollahi@kmu.ac.ir [Department of Anatomy, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Neuroscience Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Afzal Research Institute, Kerman (Iran, Islamic Republic of); Ansari, Mehdi, E-mail: mansari@kmu.ac.ir [Pharmaceutics Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Pharmaceutics Research Centre, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of)

    2016-03-15

    Magnetic nanoparticles have been used as effective vehicles for the targeted delivery of therapeutic agents that can be controlled in their concentration and distribution to a desired part of the body by using externally driven magnets. This study focuses on the synthesis, characterization, and functionalization of pullulan–spermine (PS) magnetic nanoparticles for medical applications. Magnetite nanopowder was produced by thermal decomposition of goethite (FeOOH) in oleic acid and 1-octadecene; pullulan–spermine was deposited on the magnetite nanoparticles in the form of pullulan–spermine clusters. EGFP-p53 plasmid was loaded on functionalized iron oleate to transfer into cells. Synthesized nanoparticles were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), vibrating sample magnetometry (VSM), and transmission electron microscopy (TEM). The encapsulation efficiency and drug loading efficiency of the nanocomplexes were tested. FTIR studies showed the presence of oleic acid and 1-octadecene in the iron oleate nanopowder and verified the interaction between spermine and pullulan. The characteristic bands of PS in the spectrum of the pullulan–spermine-coated iron oleate (PSCFO) confirmed that PS covered the surface of the iron oleate particles. TEM studies showed the average size of the iron oleate nanopowder, the PSCFO, and the plasmid-carrying PSCFO (PSCFO/pEGFP-p53) to be 34±12 nm, 100±50 nm and 172±3 nm, respectively. Magnetic measurements revealed that magnetic saturation of the PSCFO was lower in comparison with the iron oleate nanopowder due to the presence of organic compounds in the former. In cytotoxicity tests performed using U87 cells as glioblastoma cells, a 92% survival rate was observed at 50 µg/µl of the plasmid-carrying PSCFO, with an IC{sub 50} value of 189 µg/µl. - Highlights: • Magnetite nanopowder was produced by thermal decomposition method. • TEM studies showed the average size of

  8. Synthesis, characterization, and cytotoxicity of the plasmid EGFP-p53 loaded on pullulan–spermine magnetic nanoparticles

    International Nuclear Information System (INIS)

    Eslaminejad, Touba; Nematollahi-Mahani, Seyed Noureddin; Ansari, Mehdi

    2016-01-01

    Magnetic nanoparticles have been used as effective vehicles for the targeted delivery of therapeutic agents that can be controlled in their concentration and distribution to a desired part of the body by using externally driven magnets. This study focuses on the synthesis, characterization, and functionalization of pullulan–spermine (PS) magnetic nanoparticles for medical applications. Magnetite nanopowder was produced by thermal decomposition of goethite (FeOOH) in oleic acid and 1-octadecene; pullulan–spermine was deposited on the magnetite nanoparticles in the form of pullulan–spermine clusters. EGFP-p53 plasmid was loaded on functionalized iron oleate to transfer into cells. Synthesized nanoparticles were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), vibrating sample magnetometry (VSM), and transmission electron microscopy (TEM). The encapsulation efficiency and drug loading efficiency of the nanocomplexes were tested. FTIR studies showed the presence of oleic acid and 1-octadecene in the iron oleate nanopowder and verified the interaction between spermine and pullulan. The characteristic bands of PS in the spectrum of the pullulan–spermine-coated iron oleate (PSCFO) confirmed that PS covered the surface of the iron oleate particles. TEM studies showed the average size of the iron oleate nanopowder, the PSCFO, and the plasmid-carrying PSCFO (PSCFO/pEGFP-p53) to be 34±12 nm, 100±50 nm and 172±3 nm, respectively. Magnetic measurements revealed that magnetic saturation of the PSCFO was lower in comparison with the iron oleate nanopowder due to the presence of organic compounds in the former. In cytotoxicity tests performed using U87 cells as glioblastoma cells, a 92% survival rate was observed at 50 µg/µl of the plasmid-carrying PSCFO, with an IC 50 value of 189 µg/µl. - Highlights: • Magnetite nanopowder was produced by thermal decomposition method. • TEM studies showed the average size of the

  9. Identification of neurons that express ghrelin receptors in autonomic pathways originating from the spinal cord.

    Science.gov (United States)

    Furness, John B; Cho, Hyun-Jung; Hunne, Billie; Hirayama, Haruko; Callaghan, Brid P; Lomax, Alan E; Brock, James A

    2012-06-01

    Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with

  10. Construction and Characterization of Insect Cell-Derived Influenza VLP: Cell Binding, Fusion, and EGFP Incorporation

    Directory of Open Access Journals (Sweden)

    Yi-Shin Pan

    2010-01-01

    Full Text Available We have constructed virus-like particles (VLPs harboring hemagglutinin (HA, neuraminidase (NA, matrix protein 1 (M1 ,and proton channel protein (M2 using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.

  11. Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

    Energy Technology Data Exchange (ETDEWEB)

    Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad, E-mail: amtamadon@gmail.com [Shiraz University of Medical Sciences, Department of Pharmaceutics, School of Pharmacy (Iran, Islamic Republic of)

    2016-05-15

    Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and {sup 1}H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (<50 µg/ml) depending on the dendrimer generation and cholesterol/amine mole ratio. Cholesterol conjugation resulted in higher resistance of the condensed plasmid DNA in a competition assay with heparin sulfate. Also, the transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol{sub 40 %}) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

  12. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    Directory of Open Access Journals (Sweden)

    Weir Jerry P

    2007-05-01

    Full Text Available Abstract Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2 BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.

  13. Characterization of Aromatase Expression in the Adult Male and Female Mouse Brain. I. Coexistence with Oestrogen Receptors α and β, and Androgen Receptors

    Science.gov (United States)

    Stanić, Davor; Dubois, Sydney; Chua, Hui Kheng; Tonge, Bruce; Rinehart, Nicole; Horne, Malcolm K.; Boon, Wah Chin

    2014-01-01

    Aromatase catalyses the last step of oestrogen synthesis. There is growing evidence that local oestrogens influence many brain regions to modulate brain development and behaviour. We examined, by immunohistochemistry, the expression of aromatase in the adult male and female mouse brain, using mice in which enhanced green fluorescent protein (EGFP) is transcribed following the physiological activation of the Cyp19A1 gene. EGFP-immunoreactive processes were distributed in many brain regions, including the bed nucleus of the stria terminalis, olfactory tubercle, medial amygdaloid nucleus and medial preoptic area, with the densest distributions of EGFP-positive cell bodies in the bed nucleus and medial amygdala. Differences between male and female mice were apparent, with the density of EGFP-positive cell bodies and fibres being lower in some brain regions of female mice, including the bed nucleus and medial amygdala. EGFP-positive cell bodies in the bed nucleus, lateral septum, medial amygdala and hypothalamus co-expressed oestrogen receptor (ER) α and β, or the androgen receptor (AR), although single-labelled EGFP-positive cells were also identified. Additionally, single-labelled ERα−, ERβ- or AR-positive cell bodies often appeared to be surrounded by EGFP-immunoreactive nerve fibres/terminals. The widespread distribution of EGFP-positive cell bodies and fibres suggests that aromatase signalling is common in the mouse brain, and that locally synthesised brain oestrogens could mediate biological effects by activating pre- and post-synaptic oestrogen α and β receptors, and androgen receptors. The higher number of EGFP-positive cells in male mice may indicate that the autocrine and paracrine effects of oestrogens are more prominent in males than females. PMID:24646567

  14. Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

    Directory of Open Access Journals (Sweden)

    Tripti Tamhane

    2015-12-01

    Full Text Available The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015 [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

  15. CNPase Expression in Olfactory Ensheathing Cells

    Directory of Open Access Journals (Sweden)

    Christine Radtke

    2011-01-01

    Full Text Available A large body of work supports the proposal that transplantation of olfactory ensheathing cells (OECs into nerve or spinal cord injuries can promote axonal regeneration and remyelination. Yet, some investigators have questioned whether the transplanted OECs associate with axons and form peripheral myelin, or if they recruit endogenous Schwann cells that form myelin. Olfactory bulbs from transgenic mice expressing the enhanced green fluorescent protein (eGFP under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase promoter were studied. CNPase is expressed in myelin-forming cells throughout their lineage. We examined CNPase expression in both in situ in the olfactory bulb and in vitro to determine if OECs express CNPase commensurate with their myelination potential. eGFP was observed in the outer nerve layer of the olfactory bulb. Dissociated OECs maintained in culture had both intense eGFP expression and CNPase immunostaining. Transplantation of OECs into transected peripheral nerve longitudinally associated with the regenerated axons. These data indicate that OECs in the outer nerve layer of the olfactory bulb of CNPase transgenic mice express CNPase. Thus, while OECs do not normally form myelin on olfactory nerve axons, their expression of CNPase is commensurate with their potential to form myelin when transplanted into injured peripheral nerve.

  16. Expression of sall4 in taste buds of zebrafish.

    Science.gov (United States)

    Jackson, Robyn; Braubach, Oliver R; Bilkey, Jessica; Zhang, Jing; Akimenko, Marie-Andrée; Fine, Alan; Croll, Roger P; Jonz, Michael G

    2013-07-01

    We characterized the expression of sall4, a gene encoding a zinc finger transcription factor involved in the maintenance of embryonic stem cells, in taste buds of zebrafish (Danio rerio). Using an enhancer trap line (ET5), we detected enhanced green fluorescent protein (EGFP) in developing and adult transgenic zebrafish in regions containing taste buds: the lips, branchial arches, and the nasal and maxillary barbels. Localization of EGFP to taste cells of the branchial arches and lips was confirmed by co-immunolabeling with antibodies against calretinin and serotonin, and a zebrafish-derived neuronal marker (zn-12). Transgenic insertion of the ET construct into the zebrafish genome was evaluated and mapped to chromosome 23 in proximity (i.e. 23 kb) to the sall4 gene. In situ hybridization and expression analysis between 24 and 96 h post-fertilization (hpf) demonstrated that transgenic egfp expression in ET5 zebrafish was correlated with the spatial and temporal pattern of expression of sall4 in the wild-type. Expression was first observed in the central nervous system and branchial arches at 24 hpf. At 48 hpf, sall4 and egfp expression was observed in taste bud primordia surrounding the mouth and branchial arches. At 72 and 96 hpf, expression was detected in the upper and lower lips and branchial arches. Double fluorescence in situ hybridization at 3 and 10 dpf confirmed colocalization of sall4 and egfp in the lips and branchial arches. These studies reveal sall4 expression in chemosensory cells and implicate this transcription factor in the development and renewal of taste epithelia in zebrafish. Copyright © 2013 Wiley Periodicals, Inc.

  17. Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7.

    Science.gov (United States)

    Roe, Andrew J; Naylor, Stuart W; Spears, Kevin J; Yull, Helen M; Dransfield, Tracy A; Oxford, Matthew; McKendrick, Iain J; Porter, Megan; Woodward, Martin J; Smith, David G E; Gally, David L

    2004-10-01

    Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.

  18. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  19. The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor-endothelial cell interactions.

    Science.gov (United States)

    Rezaei, Maryam; Cao, Jiahui; Friedrich, Katrin; Kemper, Björn; Brendel, Oliver; Grosser, Marianne; Adrian, Manuela; Baretton, Gustavo; Breier, Georg; Schnittler, Hans-Joachim

    2018-01-01

    The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased β-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.

  20. Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Tanja B; Mathiesen, Christian K

    2011-01-01

    to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log(10) focus-forming units (FFU)/ml. 2a(J6) with ¿40 or ¿25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFP¿40 acquired various...... deletions in EGFP, while 2a(J6)¿40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5A¿40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those...

  1. EXPRESS

    International Nuclear Information System (INIS)

    Ancelin, C.; Le, P.; DeSaint-Quentin, S.; Villatte, N.

    1987-01-01

    This paper presents EXPRESS, an expert system developed for the automation of reliability studies. The first part consists in the description of the method for static thermohydraulic systems. In this step, the authors define the knowledge representation based on the two inference engines - ALOUETTE and LCR developed by EDF. They explain all the process to construct a fault tree from a topological and functional description of the system. Numerous examples are exhibited in illustration of the method. This is followed by the lessons derived from the studies performed on some safety systems of the PALUEL nuclear plant. The development of the same approach for electric power systems is described, insisting on the difference resulting from the sequential nature of these systems. Finally, they show the main advantages identified during the studies

  2. Comparison of expression vectors in Lactobacillus reuteri strains.

    Science.gov (United States)

    Lizier, Michela; Sarra, Pier G; Cauda, Roberto; Lucchini, Franco

    2010-07-01

    The synthesis of heterologous proteins in lactobacilli is strongly influenced by the promoter selected for the expression. In addition, the activity of the promoters themselves may vary among different bacterial hosts. Three different promoters were investigated for their capability to drive enhanced green fluorescent protein (EGFP) expression in Lactococcus lactis spp. cremoris MG1363, in Lactobacillus reuteri DSM 20016(T) and in five L. reuteri strains isolated from chicken crops. The promoters of the Lactobacillus acidophilus surface layer protein gene (slp), L. acidophilus lactate dehydrogenase gene (ldhL) and enterococcal rRNA adenine N-6-methyltransferase gene (ermB) were fused to the coding sequence of EGFP and inserted into the backbone of the pTRKH3 shuttle vector (pTRKH3-slpGFP, pTRKH3-ldhGFP, pTRKH3-ermGFP). Besides conventional analytical methods, a new quick fluorimetric approach was set up to quantify the EGFP fluorescence in transformed clones using the Qubit() fluorometer. ermB proved to be the most effective promoter in L. reuteri isolates, producing 3.90 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Under the same conditions, the ldhL promoter produced 2.66 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Even though the slp promoter was efficient in L. lactis spp. cremoris MG1363, it was nearly inactive both in L. reuteri DSM 20016(T) and in L. reuteri isolates.

  3. DNMT 1 maintains hypermethylation of CAG promoter specific region and prevents expression of exogenous gene in fat-1 transgenic sheep.

    Science.gov (United States)

    Yang, Chunrong; Shang, Xueying; Cheng, Lei; Yang, Lei; Liu, Xuefei; Bai, Chunling; Wei, Zhuying; Hua, Jinlian; Li, Guangpeng

    2017-01-01

    Methylation is an important issue in gene expression regulation and also in the fields of genetics and reproduction. In this study, we created fat-1 transgenic sheep, investigated the fine-mapping and the modulatory mechanisms of promoter methylation. Sheep fetal fibroblasts were transfected by pCAG-fat1-IRES-EGFP. Monoclonal cell line was screened as nuclear donor and carried out nuclear transfer (441 transgenic cloned embryos, 52 synchronism recipient sheep). Six offsprings were obtained. Expressions of exogenous genes fat-1 and EGFP were detectable in 10 examined tissues and upregulated omega-3 fatty acid content. Interestingly, more or less EGFP negative cells were detectable in the positive transgenic fetal skin cells. EGFP negative and positive cells were sorted by flow cytometry, and their methylation status in the whole promoter region (1701 nt) were investigated by bisulphate sequencing. The fine-mapping of methylation in CAG promoter were proposed. The results suggested that exogenous gene expression was determined by the methylation status from 721-1346 nt and modulated by methylation levels at 101, 108 and 115 nt sites in CAG promoter. To clarify the regulatory mechanism of methylation, examination of four DNA methyltransferases (DNMTs) demonstrated that hypermethylation of CAG promoter is mainly maintained by DNMT 1 in EGFP negative cells. Furthermore, investigation of the cell surface antigen CD34, CD45 and CD166 indicated that EGFP positive and negative cells belong to different types. The present study systematically clarified methylation status of CAG promoter in transgenic sheep and regulatory mechanism, which will provide research strategies for gene expression regulation in transgenic animals.

  4. Expression of cDNAs in human Natural Killer cell lines by retroviral transduction.

    Science.gov (United States)

    Miah, S M Shahjahan; Campbell, Kerry S

    2010-01-01

    Human NK-like cell lines are difficult to transfect using standard mammalian expression vectors and conventional transfection protocols, but they are susceptible to retroviral transduction as a means to introduce cDNAs. Our laboratory has exploited this technique to study a number of receptors in human NK cell lines. The method utilizes a bicistronic retroviral vector that co-expresses either drug resistance or enhanced green fluorescent protein (EGFP) in parallel with the gene of interest. After a single infection with recombinant retrovirus, transduced NK cells can be sorted for expression of EGFP or the transduced cell surface marker. Alternatively, cells expressing the transduced cDNAs can be selected for by treatment with neomycin, puromycin, or hygromycin. Using this method, the sorted/selected cells uniformly express the gene of interest and the expression is stable for many weeks of culture.

  5. Transcriptional Profiling of Cholinergic Neurons From Basal Forebrain Identifies Changes in Expression of Genes Between Sleep and Wake.

    Science.gov (United States)

    Nikonova, Elena V; Gilliland, Jason DA; Tanis, Keith Q; Podtelezhnikov, Alexei A; Rigby, Alison M; Galante, Raymond J; Finney, Eva M; Stone, David J; Renger, John J; Pack, Allan I; Winrow, Christopher J

    2017-06-01

    To assess differences in gene expression in cholinergic basal forebrain cells between sleeping and sleep-deprived mice sacrificed at the same time of day. Tg(ChAT-eGFP)86Gsat mice expressing enhanced green fluorescent protein (eGFP) under control of the choline acetyltransferase (Chat) promoter were utilized to guide laser capture of cholinergic cells in basal forebrain. Messenger RNA expression levels in these cells were profiled using microarrays. Gene expression in eGFP(+) neurons was compared (1) to that in eGFP(-) neurons and to adjacent white matter, (2) between 7:00 am (lights on) and 7:00 pm (lights off), (3) between sleep-deprived and sleeping animals at 0, 3, 6, and 9 hours from lights on. There was a marked enrichment of ChAT and other markers of cholinergic neurons in eGFP(+) cells. Comparison of gene expression in these eGFP(+) neurons between 7:00 am and 7:00 pm revealed expected differences in the expression of clock genes (Arntl2, Per1, Per2, Dbp, Nr1d1) as well as mGluR3. Comparison of expression between spontaneous sleep and sleep-deprived groups sacrificed at the same time of day revealed a number of transcripts (n = 55) that had higher expression in sleep deprivation compared to sleep. Genes upregulated in sleep deprivation predominantly were from the protein folding pathway (25 transcripts, including chaperones). Among 42 transcripts upregulated in sleep was the cold-inducible RNA-binding protein. Cholinergic cell signatures were characterized. Whether the identified genes are changing as a consequence of differences in behavioral state or as part of the molecular regulatory mechanism remains to be determined. © Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

  6. Retinal Astrocytes and GABAergic Wide-Field Amacrine Cells Express PDGFRα: Connection to Retinal Ganglion Cell Neuroprotection by PDGF-AA.

    Science.gov (United States)

    Takahama, Shokichi; Adetunji, Modupe O; Zhao, Tantai; Chen, Shan; Li, Wei; Tomarev, Stanislav I

    2017-09-01

    Our previous experiments demonstrated that intravitreal injection of platelet-derived growth factor-AA (PDGF-AA) provides retinal ganglion cell (RGC) neuroprotection in a rodent model of glaucoma. Here we used PDGFRα-enhanced green fluorescent protein (EGFP) mice to identify retinal cells that may be essential for RGC protection by PDGF-AA. PDGFRα-EGFP mice expressing nuclear-targeted EGFP under the control of the PDGFRα promoter were used. Localization of PDGFRα in the neural retina was investigated by confocal imaging of EGFP fluorescence and immunofluorescent labeling with a panel of antibodies recognizing different retinal cell types. Primary cultures of mouse RGCs were produced by immunopanning. Neurobiotin injection of amacrine cells in a flat-mounted retina was used for the identification of EGFP-positive amacrine cells in the inner nuclear layer. In the mouse neural retina, PDGFRα was preferentially localized in the ganglion cell and inner nuclear layers. Immunostaining of the retina demonstrated that astrocytes in the ganglion cell layer and a subpopulation of amacrine cells in the inner nuclear layer express PDGFRα, whereas RGCs (in vivo or in vitro) did not. PDGFRα-positive amacrine cells are likely to be Type 45 gamma-aminobutyric acidergic (GABAergic) wide-field amacrine cells. These data indicate that the neuroprotective effect of PDGF-AA in a rodent model of glaucoma could be mediated by astrocytes and/or a subpopulation of amacrine cells. We suggest that after intravitreal injection of PDGF-AA, these cells secrete factors protecting RGCs.

  7. Expression of heterologous genes from an IRES translational cassette in replication-competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Jespersen, T.; Duch, M.; Carrasco, M.L.

    1999-01-01

    of spliced env mRNA for the SL3-3 derived vector relative to the Akv derived vectors, seemingly contributing to its low replication capacity. The EGFP expressing Akv-MLV was genetically stable for multiple rounds of infection; marker-cassette deletion revertants appeared after several replication rounds...

  8. Eicosapentenoic Acid Attenuates Allograft Rejection in an HLA-B27/EGFP Transgenic Rat Cardiac Transplantation Model.

    Science.gov (United States)

    Liu, Zhong; Hatayama, Naoyuki; Xie, Lin; Kato, Ken; Zhu, Ping; Ochiya, Takahiro; Nagahara, Yukitoshi; Hu, Xiang; Li, Xiao-Kang

    2012-01-01

    The development of an animal model bearing definite antigens is important to facilitate the evaluation and modulation of specific allo-antigen responses after transplantation. In the present study, heterotopic cardiac transplantation was performed from F344/EGFPTg and F344/HLA-B27Tg rats to F344 rats. The F344 recipients accepted the F344/EGFPTg transplants, whereas they rejected the cardiac tissue from the F344/HLA-B27Tg rats by 39.4 ± 6.5 days, due to high production of anti-HLA-B27 IgM- and IgG-specific antibodies. In addition, immunization of F344 rats with skin grafts from F344/HLA-B27Tg rats resulted in robust production of anti- HLA-B27 IgM and IgG antibodies and accelerated the rejection of a secondary cardiac allograft (7.4 ± 1.9 days). Of interest, the F344 recipients rejected cardiac grafts from double transgenic F344/HLA-B27&EGFPTg rats within 9.0 ± 3.2 days, and this was associated with a significant increase in the infiltration of lymphocytes by day 7, suggesting a role for cellular immune rejection. Eicosapentenoic acid (EPA), one of the ω-3 polyunsaturated fatty acids in fish oil, could attenuate the production of anti-HLA IgG antibodies and B-cell proliferation, significantly prolonging double transgenic F344HLA-B27&EGFPTg to F344 rat cardiac allograft survival (36.1 ± 13.6 days). Moreover, the mRNA expression in the grafts was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), revealing an increase in the expression of the HO-1, IL-10, TGF-β, IDO, and Foxp3 genes in the EPA-treated group. Hence, our data indicate that HLA-B27 and/or GFP transgenic proteins are useful for establishing a unique animal transplantation model to clarify the mechanism underlying the allogeneic cellular and humoral immune response, in which the transplant antigens are specifically presented. Furthermore, we also demonstrated that EPA was effective in the treatment of rat cardiac allograft rejection and may allow the development of

  9. Molecular and functional characterization of GAD67-expressing, newborn granule cells in mouse dentate gyrus

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    Carolina eCabezas

    2013-04-01

    Full Text Available Dentate gyrus granule cells (GCs have been suggested to synthesize both GABA and glutamate immediately after birth and under pathological conditions in the adult. Expression of the GABA synthesizing enzyme GAD67 by GCs during the first few weeks of postnatal development may then allow for transient GABA synthesis and synaptic release from these cells. Here, using the GAD67-EGFP transgenic strain G42, we explored the phenotype of GAD67-expressing GCs in the mouse dentate gyrus. We report a transient, GAD67-driven EGFP expression in differentiating GCs throughout ontogenesis. EGFP expression correlates with the expression of GAD and molecular markers of GABA release and uptake in 2-4 weeks postmitotic GCs. These rather immature cells are able to fire action potentials and are synaptically integrated in the hippocampal network. Yet they show physiological properties that differentiate them from mature GCs. Finally, GAD67-expressing GCs express a specific complement of GABAA receptor subunits as well as distinctive features of synaptic and tonic GABA signaling. Our results reveal that GAD67 expression in dentate gyrus granule cells is a transient marker of late differentiation that persists throughout life and the G42 strain may be used to visualize newborn GCs at a specific, well-defined differentiation stage.

  10. Cloning and Expression of Luteinizing Hormone Subunits in Chinese Hamster Ovary Cell Line

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    Zeinab Soleimanifar

    2016-10-01

    Full Text Available Background: Luteinizing hormone (LH was secreted by the stimulating cells of the testes and ovaries in the anterior pituitary gland. The application of this hormone is in the treatment of men and women with infertility and amenorrhea respectively.Materials and Methods: In the present study the alpha and beta subunits of human LH gene were cloned into the pEGFP-N1 expression vector and produced the recombinant LH hormone in Chinese hamster ovary (CHO eukaryotic system.Results: Alpha and beta subunits of LH hormone were cloned between NheI and BamHI cut sites of pEGFP_N1 expression plasmid and confirmed by PCR.  Hormone expression was evaluated in CHO cell line by Western blotting using the specific antibody.Conclusion: Alpha and beta subunits of LH hormone were expressed in CHO cell line perfectly.

  11. Distinct projection targets define subpopulations of mouse brainstem vagal neurons that express the autism-associated MET receptor tyrosine kinase.

    Science.gov (United States)

    Kamitakahara, Anna; Wu, Hsiao-Huei; Levitt, Pat

    2017-12-15

    Detailed anatomical tracing and mapping of the viscerotopic organization of the vagal motor nuclei has provided insight into autonomic function in health and disease. To further define specific cellular identities, we paired information based on visceral connectivity with a cell-type specific marker of a subpopulation of neurons in the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (nAmb) that express the autism-associated MET receptor tyrosine kinase. As gastrointestinal disturbances are common in children with autism spectrum disorder (ASD), we sought to define the relationship between MET-expressing (MET+) neurons in the DMV and nAmb, and the gastrointestinal tract. Using wholemount tissue staining and clearing, or retrograde tracing in a MET EGFP transgenic mouse, we identify three novel subpopulations of EGFP+ vagal brainstem neurons: (a) EGFP+ neurons in the nAmb projecting to the esophagus or laryngeal muscles, (b) EGFP+ neurons in the medial DMV projecting to the stomach, and (b) EGFP+ neurons in the lateral DMV projecting to the cecum and/or proximal colon. Expression of the MET ligand, hepatocyte growth factor (HGF), by tissues innervated by vagal motor neurons during fetal development reveal potential sites of HGF-MET interaction. Furthermore, similar cellular expression patterns of MET in the brainstem of both the mouse and nonhuman primate suggests that MET expression at these sites is evolutionarily conserved. Together, the data suggest that MET+ neurons in the brainstem vagal motor nuclei are anatomically positioned to regulate distinct portions of the gastrointestinal tract, with implications for the pathophysiology of gastrointestinal comorbidities of ASD. © 2017 Wiley Periodicals, Inc.

  12. Small animal PET imaging of HSV1-tk gene expression with {sup 124}IVDU in liver by the hydrodynamic injection

    Energy Technology Data Exchange (ETDEWEB)

    Song, I. H.; Lee, T. S.; Woo, S. G.; Jeong, J. H.; Kang, J. H.; Kim, K. M.; Chun, K. J.; Choi, C. W.; Lim, S. M. [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2007-07-01

    The liver is an important target organ for gene transfer due to its capacity for synthesizing serum protein and its involvement in numerous genetic diseases. High level of foreign gene expression in liver can be achieved by a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), so called hydrodynamic injection. This study is aimed to evaluate liver specific-gene expression of herpes simplex virus type 1 thymidine kinase(HSV1-tk) by hydrodynamic injection and image HSV1-tk expression using {sup 124}IVDU-PET. We constructed herpes simplex virus type 1 thymidine kinase (HSV1-tk)-expressing pDNA (pHSV1-tk) modified from pEGFP-N1. Hydrodynamic injection was performed using 40 {mu}g of plasmid (pEGFP/N1 or pHSV1-tk) in 2 ml of 0.85% saline solution for 20{approx}22g mice in 5 seconds intravenously. At 1 d post-hydrodynamic injection, biodistribution study was performed at 2 h post-injection of radiolabeled IVDU, fluorescence image was obtained using optical imager and small animal PET image was acquired with {sup 124}IVDU at 2 h post-injection. After PET imaging, digital whole body autoradiography (DWBA) was performed. Expression of HSV1-tk and EGFP was confirmed by RT-PCR in each liver tissue. In liver of pHSV1-tk and pEGFP/N1 injection groups, {sup 123}IVDU uptake was 5.65%ID/g and 0.98%ID/g, respectively. {sup 123}IVDU uptake in liver of pHSV1-tk injection group showed 5.7-fold higher than that of pEGFP/N1 injection group (p<0.01). On the other hand, the liver of pEGFP/N1 injection group showed fluorescence activity. In small animal PET images, {sup 124}IVDU uptake was selectively localized in liver of pHSV1-tk injection group and also checked in DWBA, but showed minimal uptake in liver of pEGFP/N1 injection mice. Hydrodynamic injection was effective to liver-specific delivery of plasmid DNA. Small animal PET image of {sup 124}IVDU could be used in the evaluation of noninvasive reporter gene imaging in liver.

  13. Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Shi Ruina [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Huang Yong [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Wang Dan [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Zhao Meiping [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Li Yuanzong [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China)]. E-mail: yzli@pku.edu.cn

    2006-09-25

    The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10{sup -8} to 2.0 x 10{sup -6} M with a detection limit of 1.6 x 10{sup -8} M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications.

  14. Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

    International Nuclear Information System (INIS)

    Shi Ruina; Huang Yong; Wang Dan; Zhao Meiping; Li Yuanzong

    2006-01-01

    The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10 -8 to 2.0 x 10 -6 M with a detection limit of 1.6 x 10 -8 M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications

  15. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

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    Tamer Z Salem

    Full Text Available The simian virus 40 polyadenylation signal (SV40 polyA has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS. In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV, the polyhedrin promoter (very late promoter transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt, and gp37. In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications.

  16. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

    Science.gov (United States)

    Salem, Tamer Z.; Seaborn, Craig P.; Turney, Colin M.; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

    2015-01-01

    The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications). PMID:26659470

  17. Members of Bitter Taste Receptor Cluster Tas2r143/Tas2r135/Tas2r126 Are Expressed in the Epithelium of Murine Airways and Other Non-gustatory Tissues

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    Shuya Liu

    2017-10-01

    Full Text Available The mouse bitter taste receptors Tas2r143, Tas2r135, and Tas2r126 are encoded by genes that cluster on chromosome 6 and have been suggested to be expressed under common regulatory elements. Previous studies indicated that the Tas2r143/Tas2r135/Tas2r126 cluster is expressed in the heart, but other organs had not been systematically analyzed. In order to investigate the expression of this bitter taste receptor gene cluster in non-gustatory tissues, we generated a BAC (bacterial artificial chromosome based transgenic mouse line, expressing CreERT2 under the control of the Tas2r143 promoter. After crossing this line with a mouse line expressing EGFP after Cre-mediated recombination, we were able to validate the Tas2r143-CreERT2 transgenic mouse line and monitor the expression of Tas2r143. EGFP-positive cells, indicating expression of members of the cluster, were found in about 47% of taste buds, and could also be found in several other organs. A population of EGFP-positive cells was identified in thymic epithelial cells, in the lamina propria of the intestine and in vascular smooth muscle cells of cardiac blood vessels. EGFP-positive cells were also identified in the epithelium of organs readily exposed to pathogens including lower airways, the gastrointestinal tract, urethra, vagina, and cervix. With respect to the function of cells expressing this bitter taste receptor cluster, RNA-seq analysis in EGFP-positive cells isolated from the epithelium of trachea and stomach showed expression of genes related to innate immunity. These data further support the concept that bitter taste receptors serve functions outside the gustatory system.

  18. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

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    Fu Juanjuan

    2011-07-01

    Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  19. Generation of knock-in mice that express nuclear enhanced green fluorescent protein and tamoxifen-inducible Cre recombinase in the notochord from Foxa2 and T loci.

    Science.gov (United States)

    Imuta, Yu; Kiyonari, Hiroshi; Jang, Chuan-Wei; Behringer, Richard R; Sasaki, Hiroshi

    2013-03-01

    The node and the notochord are important embryonic signaling centers that control embryonic pattern formation. Notochord progenitor cells present in the node and later in the posterior end of the notochord move anteriorly to generate the notochord. To understand the dynamics of cell movement during notochord development and the molecular mechanisms controlling this event, analyses of cell movements using time-lapse imaging and conditional manipulation of gene activities are required. To achieve this goal, we generated two knock-in mouse lines that simultaneously express nuclear enhanced green fluorescent protein (EGFP) and tamoxifen-inducible Cre, CreER(T2) , from two notochord gene loci, Foxa2 and T (Brachury). In Foxa2(nEGFP-CreERT2/+) and T(nEGFP-CreERT2/+) embryos, nuclei of the Foxa2 or T-expressing cells, which include the node, notochord, and endoderm (Foxa2) or wide range of posterior mesoderm (T), were labeled with EGFP at intensities that can be used for live imaging. Cre activity was also induced in cells expressing Foxa2 and T 1 day after tamoxifen administration. These mice are expected to be useful tools for analyzing the mechanisms of notochord development. Copyright © 2013 Wiley Periodicals, Inc.

  20. High yield expression and purification of equilibrative nucleoside transporter 7 (ENT7) from Arabidopsis thaliana.

    Science.gov (United States)

    Girke, Christopher; Arutyunova, Elena; Syed, Maria; Traub, Michaela; Möhlmann, Torsten; Lemieux, M Joanne

    2015-09-01

    Equilibrative nucleoside transporters (ENTs) facilitate the import of nucleosides and their analogs into cells in a bidirectional, non-concentrative manner. However, in contrast to their name, most characterized plant ENTs act in a concentrative manner. A direct characterization of any ENT protein has been hindered due to difficulties in overexpression and obtaining pure recombinant protein. The equilibrative nucleoside transporter 7 from Arabidopsis thaliana (AtENT7) was expressed in Xenopus laevis oocytes to assess mechanism of substrate uptake. Recombinant protein fused to enhanced green fluorescent protein (eGFP) was expressed in Pichia pastoris to characterize its oligomeric state by gel filtration and substrate binding by microscale thermophoresis (MST). AtENT7 expressed in X. laevis oocytes works as a classic equilibrative transporter. The expression of AtENT7-eGFP in the P. pastoris system yielded milligram amounts of pure protein that exists as stable homodimers. The concentration dependent binding of purine and pyrimidine nucleosides to the purified recombinant protein, assessed by MST, confirmed that AtENT7-eGFP is properly folded. For the first time the binding of nucleobases was observed for AtENT7. The availability of pure recombinant AtENT7 will permit detailed kinetic and structural studies of this unique member of the ENT family and, given the functional similarity to mammalian ENTs, will serve as a good model for understanding the structural basis of translocation mechanism for the family. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. The stealth episome: suppression of gene expression on the excised genomic island PPHGI-1 from Pseudomonas syringae pv. phaseolicola.

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    Scott A C Godfrey

    2011-03-01

    Full Text Available Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1, which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1, revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state.

  2. Quantitative analysis of lentiviral transgene expression in mice over seven generations.

    Science.gov (United States)

    Wang, Yong; Song, Yong-tao; Liu, Qin; Liu, Cang'e; Wang, Lu-lu; Liu, Yu; Zhou, Xiao-yang; Wu, Jun; Wei, Hong

    2010-10-01

    Lentiviral transgenesis is now recognized as an extremely efficient and cost-effective method to produce transgenic animals. Transgenes delivered by lentiviral vectors exhibited inheritable expression in many species including those which are refractory to genetic modification such as non-human primates. However, epigenetic modification was frequently observed in lentiviral integrants, and transgene expression found to be inversely correlated with methylation density. Recent data showed that about one-third lentiviral integrants exhibited hypermethylation and low expression, but did not demonstrate whether those integrants with high expression could remain constant expression and hypomethylated during long term germline transmission. In this study, using lentiviral eGFP transgenic mice as the experimental animals, lentiviral eGFP expression levels and its integrant numbers in genome were quantitatively analyzed by fluorescent quantitative polymerase-chain reaction (FQ-PCR), using the house-keeping gene ribosomal protein S18 (Rps18) and the single copy gene fatty acid binding protein of the intestine (Fabpi) as the internal controls respectively. The methylation densities of the integrants were quantitatively analyzed by bisulfite sequencing. We found that the lentiviral integrants with high expression exhibited a relative constant expression level per integrant over at least seven generations. Besides, the individuals containing these integrants exhibited eGFP expression levels which were positively and almost linearly correlated with the integrant numbers in their genomes, suggesting that no remarkable position effect on transgene expression of the integrants analyzed was observed. In addition, over seven generations the methylation density of these integrants did not increase, but rather decreased remarkably, indicating that these high expressing integrants were not subjected to de novo methylation during at least seven generations of germline transmission. Taken

  3. Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals.

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    Yeon Sook Cho

    Full Text Available Interleukin-7 (IL-7 is a cytokine essential for T cell homeostasis, and is clinically important. However, the regulatory mechanism of IL-7 gene expression is not well known, and a systematic approach to screen chemicals that regulate IL-7 expression has not yet been developed. In this study, we attempted to develop human reporter cell lines using CRISPR/Cas9-mediated genome editing technology. For this purpose, we designed donor DNA that contains an enhanced green fluorescent protein (eGFP gene, drug selection cassette, and modified homologous arms which are considered to enhance the translation of the eGFP reporter transcript, and also a highly efficient single-guide RNA with a minimal off-target effect to target the IL-7 start codon region. By applying this system, we established IL-7 eGFP reporter cell lines that could report IL-7 gene transcription based on the eGFP protein signal. Furthermore, we utilized the cells to run a pilot screen campaign for IL-7-upregulating chemicals in a high-throughput format, and identified a chemical that can up-regulate IL-7 gene transcription. Collectively, these results suggest that our IL-7 reporter system can be utilized in large-scale chemical library screening to reveal novel IL-7 regulatory pathways and to identify potential drugs for development of new treatments in immunodeficiency disease.

  4. Anti-tumor effect of a recombinant plasmid expressing human interleukin-12: an initial research

    International Nuclear Information System (INIS)

    Zheng Chuansheng; Xia Xiangwen; Feng Gansheng; Li Xin; Liang Huimin; Liang Bin

    2010-01-01

    Objective: To study the anti-tumor effect of a recombinant plasmid expressing human interleukin-12 (pEGFP-CI I L- 12) in vivo and in vitro. Methods: We transduct the recombinant gene (pEGFP-CI I L-12) to liver cancer cell HepG 2 in vitro, and detect reproductive activity of the cell using MTT and the activity of expressing vascular endothelial growth factor(VEGF) using semiquantitative PCR. And then, we deliver the gene to rabbit liver tumor tissue intraarterial and combine with chemoembolization to observe the anti- tumor effect to VX 2 tumor in vivo. Results: There are no statistical difference compared With control group in activity of reproductive and expressing VEGF in vitro. In vivo, tumor growth rate significantly reduce in gene therapy combined with chemoembolization group. Conclusion: Recombinant gene (pEGFP-Cl I L-12) exhibit significant anti-tumor effect in vivo but not in vitro, perhaps the anti-tumor effect is associated with an indirect pathway instead of a direct pathway. (authors)

  5. GFP expression by intracellular gene delivery of GFP-coding fragments using nanocrystal quantum dots

    International Nuclear Information System (INIS)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Fujioka, Kouki; Hanada, Sanshiro; Yamamoto, Kenji; Yasuhara, Masato; Kondo, Akihiko

    2008-01-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

  6. Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

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    Mandy Y M Lo

    Full Text Available Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

  7. 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.

  8. Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

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    Shaoxing YANG

    2012-12-01

    Full Text Available Background and objective CD147, a type of transmembrane glycoprotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD147 gene was amplified by real-time polymerase chain reaction (RT-PCR, inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable, overexpressed cell line named A549-CD147. The mRNA expression of MMP-9 was examined by RT-PCR. The proliferation and invasive ability of the human lung cancer cells before and after transfection were examined by the CCK-8 and Transwell methods. Results A CD147 lentiviral expression vector (pEGFP-CD147 was successfully constructed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blot analyses revealed increased mRNA and protein expression of CD147 gene in cells transfected with pEGFP-CD147 compared with the control groups. Therefore, the A549-CD147 cell line was successfully established through the experiment. The mRNA expression of MMP-9 also significantly increased after the upregulation of CD147 expression. Meanwhile, CCK-8 and Transwell assays indicated that the proliferation and invasive ability significantly increased in the A549-CD147 cells. Conclusion A lentiviral CD147 expression vector and its A549 cell line (A549-CD14 were successfully constructed. CD147 overexpression upregulated the protein expression of MMP-9, and strengthened the proliferation and invasive ability of human lung adenocarcinoma cells.

  9. Expression of gastrin-releasing peptide by excitatory interneurons in the mouse superficial dorsal horn.

    Science.gov (United States)

    Gutierrez-Mecinas, Maria; Watanabe, Masahiko; Todd, Andrew J

    2014-12-11

    Gastrin-releasing peptide (GRP) and its receptor have been shown to play an important role in the sensation of itch. However, although GRP immunoreactivity has been detected in the spinal dorsal horn, there is debate about whether this originates from primary afferents or local excitatory interneurons. We therefore examined the relation of GRP immunoreactivity to that seen with antibodies that label primary afferent or excitatory interneuron terminals. We tested the specificity of the GRP antibody by preincubating with peptides with which it could potentially cross-react. We also examined tissue from a mouse line in which enhanced green fluorescent protein (EGFP) is expressed under control of the GRP promoter. GRP immunoreactivity was seen in both primary afferent and non-primary glutamatergic axon terminals in the superficial dorsal horn. However, immunostaining was blocked by pre-incubation of the antibody with substance P, which is present at high levels in many nociceptive primary afferents. EGFP+ cells in the GRP-EGFP mouse did not express Pax2, and their axons contained the vesicular glutamate transporter 2 (VGLUT2), indicating that they are excitatory interneurons. In most cases, their axons were also GRP-immunoreactive. Multiple-labelling immunocytochemical studies indicated that these cells did not express either of the preprotachykinin peptides, and that they generally lacked protein kinase Cγ, which is expressed by a subset of the excitatory interneurons in this region. These results show that GRP is expressed by a distinct population of excitatory interneurons in laminae I-II that are likely to be involved in the itch pathway. They also suggest that the GRP immunoreactivity seen in primary afferents in previous studies may have resulted from cross-reaction of the GRP antibody with substance P or the closely related peptide neurokinin A.

  10. Fluorescent reporter signals, EGFP and DsRed, encoded in HIV-1 facilitate the detection of productively infected cells and cell-associated viral replication levels

    Directory of Open Access Journals (Sweden)

    Kazutaka eTerahara

    2012-01-01

    Full Text Available Flow cytometric analysis is a reliable and convenient method for investigating molecules at the single cell level. Previously, recombinant human immunodeficiency virus type 1 (HIV-1 strains were constructed that express a fluorescent reporter, either enhanced green fluorescent protein or DsRed, which allow the monitoring of HIV-1-infected cells by flow cytometry. The present study further investigated the potential of these recombinant viruses in terms of whether the HIV-1 fluorescent reporters would be helpful in evaluating viral replication based on fluorescence intensity. When primary CD4+ T cells were infected with recombinant viruses, the fluorescent reporter intensity measured by flow cytometry was associated with the level of CD4 downmodulation and Gag p24 expression in infected cells. Interestingly, some HIV-1-infected cells, in which CD4 was only moderately downmodulated, were reporter-positive but Gag p24-negative. Furthermore, when the activation status of primary CD4+ T cells was modulated by T cell receptor-mediated stimulation, we confirmed the preferential viral production upon strong stimulation and showed that the intensity of the fluorescent reporter within a proportion of HIV-1-infected cells was correlated with the viral replication level. These findings indicate that a fluorescent reporter encoded within HIV-1 is useful for the sensitive detection of productively-infected cells at different stages of infection and for evaluating cell-associated viral replication at the single cell level.

  11. Regeneration of alveolar type I and II cells from Scgb1a1-expressing cells following severe pulmonary damage induced by bleomycin and influenza.

    Directory of Open Access Journals (Sweden)

    Dahai Zheng

    Full Text Available The lung comprises an extensive surface of epithelia constantly exposed to environmental insults. Maintaining the integrity of the alveolar epithelia is critical for lung function and gaseous exchange. However, following severe pulmonary damage, what progenitor cells give rise to alveolar type I and II cells during the regeneration of alveolar epithelia has not been fully determined. In this study, we have investigated this issue by using transgenic mice in which Scgb1a1-expressing cells and their progeny can be genetically labeled with EGFP. We show that following severe alveolar damage induced either by bleomycin or by infection with influenza virus, the majority of the newly generated alveolar type II cells in the damaged parenchyma were labeled with EGFP. A large proportion of EGFP-expressing type I cells were also observed among the type II cells. These findings strongly suggest that Scgb1a1-expressing cells, most likely Clara cells, are a major cell type that gives rise to alveolar type I and II cells during the regeneration of alveolar epithelia in response to severe pulmonary damage in mice.

  12. Comparative immunological evaluation of recombinant Salmonella Typhimurium strains expressing model antigens as live oral vaccines.

    Science.gov (United States)

    Zheng, Song-yue; Yu, Bin; Zhang, Ke; Chen, Min; Hua, Yan-Hong; Yuan, Shuofeng; Watt, Rory M; Zheng, Bo-Jian; Yuen, Kwok-Yung; Huang, Jian-Dong

    2012-09-26

    Despite the development of various systems to generate live recombinant Salmonella Typhimurium vaccine strains, little work has been performed to systematically evaluate and compare their relative immunogenicity. Such information would provide invaluable guidance for the future rational design of live recombinant Salmonella oral vaccines. To compare vaccine strains encoded with different antigen delivery and expression strategies, a series of recombinant Salmonella Typhimurium strains were constructed that expressed either the enhanced green fluorescent protein (EGFP) or a fragment of the hemagglutinin (HA) protein from the H5N1 influenza virus, as model antigens. The antigens were expressed from the chromosome, from high or low-copy plasmids, or encoded on a eukaryotic expression plasmid. Antigens were targeted for expression in either the cytoplasm or the outer membrane. Combinations of strategies were employed to evaluate the efficacy of combined delivery/expression approaches. After investigating in vitro and in vivo antigen expression, growth and infection abilities; the immunogenicity of the constructed recombinant Salmonella strains was evaluated in mice. Using the soluble model antigen EGFP, our results indicated that vaccine strains with high and stable antigen expression exhibited high B cell responses, whilst eukaryotic expression or colonization with good construct stability was critical for T cell responses. For the insoluble model antigen HA, an outer membrane expression strategy induced better B cell and T cell responses than a cytoplasmic strategy. Most notably, the combination of two different expression strategies did not increase the immune response elicited. Through systematically evaluating and comparing the immunogenicity of the constructed recombinant Salmonella strains in mice, we identified their respective advantages and deleterious or synergistic effects. Different construction strategies were optimally-required for soluble versus

  13. Imaging of Herpes Simplex Virus Type 1 Thymidine Kinase Gene Expression with Radiolabeled 5-(2-iodovinyl)-2'-deoxyuridine (IVDU) in Liver by Hydrodynamic-based Procedure

    Energy Technology Data Exchange (ETDEWEB)

    Song, In Ho; Lee, Tae Sup; Kang, Joo Hyun; Lee, Yong Jin; Kim, Kwang Il; An, Gwang Il; Chung, Wee Sup; Cheon, Gi Jeong; Choi, Chang Woon; Lim, Sang Moo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2009-10-15

    Hydrodynamic-based procedure is a simple and effective gene delivery method to lead a high gene expression in liver tissue. Non-invasive imaging reporter gene system has been used widely with herpes simplex virus type 1 thymidine kinase (HSV1-tk) and its various substrates. In the present study, we investigated to image the expression of HSV1-tk gene with 5-(2-iodovinyl)-2'-deoxyuridine (IVDU) in mouse liver by the hydrodynamicbased procedure. HSV1-tk or enhanced green fluorescence protein (EGFP) encoded plasmid DNA was transferred into the mouse liver by hydrodynamic injection. At 24 h post-injection, RT-PCR, biodistribution, fluorescence imaging, nuclear imaging and digital wholebody autoradiography (DWBA) were performed to confirm transferred gene expression. In RT-PCR assay using mRNA from the mouse liver, specific bands of HSV1-tk and EGFP gene were observed in HSV1-tk and EGFP expressing plasmid injected mouse, respectively. Higher uptake of radiolabeled IVDU was exhibited in liver of HSV1-tk gene transferred mouse by biodistribution study. In fluorescence imaging, the liver showed specific fluorescence signal in EGFP gene transferred mouse. Gamma-camera image and DWBA results showed that radiolabeled IVDU was accumulated in the liver of HSV1-tk gene transferred mouse. In this study, hydrodynamic-based procedure was effective in liver-specific gene delivery and it could be quantified with molecular imaging methods. Therefore, co-expression of HSV1-tk reporter gene and target gene by hydrodynamic-based procedure is expected to be a useful method for the evaluation of the target gene expression level with radiolabeled IVDU.

  14. Defining POMC neurons using transgenic reagents: impact of transient Pomc expression in diverse immature neuronal populations.

    Science.gov (United States)

    Padilla, Stephanie L; Reef, Daniel; Zeltser, Lori M

    2012-03-01

    Melanocortin signaling plays a central role in the regulation of phenotypes related to body weight and energy homeostasis. To specifically target and study the function of proopiomelanocortin (POMC) neurons, Pomc promoter elements have been utilized to generate reporter and Cre recombinase transgenic reagents. Across gestation, we find that Pomc is dynamically expressed in many sites in the developing mouse forebrain, midbrain, hindbrain, spinal cord, and retina. Although Pomc expression in most embryonic brain regions is transient, it is sufficient to direct Cre-mediated recombination of floxed alleles. We visualize the populations affected by this transgene by crossing Pomc-Cre mice to ROSA reporter strains and identify 62 sites of recombination throughout the adult brain, including several nuclei implicated in energy homeostasis regulation. To compare the relationship between acute Pomc promoter activity and Pomc-Cre-mediated recombination at the single cell level, we crossed Pomc-enhanced green fluorescent protein (eGFP) and Pomc-Cre;ROSA-tdTomato lines. We detect the highest concentration of Pomc-eGFP+ cells in the arcuate nucleus of the hypothalamus and dentate gyrus but also observe smaller populations of labeled cells in the nucleus of the solitary tract, periventricular zone of the third ventricle, and cerebellum. Consistent with the dynamic nature of Pomc expression in the embryo, the vast majority of neurons marked with the tdTomato reporter do not express eGFP in the adult. Thus, recombination in off-target sites could contribute to physiological phenotypes using Pomc-Cre transgenics. For example, we find that approximately 83% of the cells in the arcuate nucleus of the hypothalamus immunoreactive for leptin-induced phosphorylated signal transducer and activator of transcription 3 are marked with Pomc-Cre;ROSA-tdTomato; only 13% of these are eGFP+ POMC neurons.

  15. Cell fusion phenomena detected after in utero transplantation of Ds-red-harboring porcine amniotic fluid stem cells into EGFP transgenic mice.

    Science.gov (United States)

    Peng, Shao-Yu; Chen, Yu-Hsu; Chou, Chih-Jen; Wang, Yao-Horng; Lee, Hung-Maan; Cheng, Winston Teng-Kui; Shaw, S W Steven; Wu, Shinn-Chih

    2014-05-01

    Amniotic fluid stem cells (AFSCs) are derived from the amniotic fluid of the developing fetus and can give rise to diverse differentiated cells of ectoderm, mesoderm, and endoderm lineages. Intrauterine transplantation is an approach used to cure inherited genetic fetal defects during the gestation period of pregnant dams. Certain disease such as osteogenesis imperfecta was successfully treated in affected fetal mice using this method. However, the donor cell destiny remains uncertain. The purpose of this study was to investigate the biodistribution and cell fate of Ds-red-harboring porcine AFSCs (Ds-red pAFSCs) after intrauterine transplantation into enhanced green fluorescent protein-harboring fetuses of pregnant mice. Pregnant mice (12.5 days) underwent open laparotomy with intrauterine pAFSC transplantation (5 × 10(4) cells per pup) into fetal peritoneal cavity. Three weeks after birth, the mice were sacrificed. Several samples from different organs were obtained for histological examination and flow cytometric analysis. Ds-red pAFSCs migrated most frequently into the intestines. Furthermore, enhanced green fluorescent protein and red fluorescent protein signals were co-expressed in the intestine and liver cells via immunohistochemistry studies. In utero xenotransplantation of pAFSCs fused with recipient intestinal cells instead of differentiating or maintaining the undifferentiated status in the tissue. © 2014 John Wiley & Sons, Ltd.

  16. [Establishment and application of a Vero cell line stably expressing raccoon dog SLAM, the cellular receptor of canine distemper virus].

    Science.gov (United States)

    Zhao, Jianjun; Yan, Ruxun; Zhang, Hailing; Zhang, Lei; Hu, Bo; Bai, Xue; Shao, Xiqun; Chai, Xiuli; Yan, Xijun; Wu, Wei

    2012-12-04

    The signaling lymphocyte activation molecule (SLAM, also known as CD150), is used as a cellular receptor by canine distemper virus (CDV). Wild-type strains of CDVs can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. Our aim is to establish a Vero cells expressing raccoon dog SLAM (rSLAM) to efficiently isolate CDV from pathological samples. A eukaryotic expression plasmid, pIRES2-EGFP-rSLAMhis, containing rSLAM gene fused with six histidine-coding sequence, EGFP gene, and neomycin resistance gene was constructed. After transfection with the plasmid, a stable cell line, Vero-rSLAM, was screened from Vero cells with the identification of EGFP reporter and G418 resistance. Three CD positive specimens from infected foxes and raccoon dogs were inoculated to Vero-rSLAM cells for CDV isolation. Foxes and raccoon dogs were inoculated subcutaneously LN (10)fl strain with 4 x 10(2.39)TCID50 dose to evaluate pathogenicity of CDV isolations. The rSLAMh fused gene was shown to transcript and express stably in Vero-rSLAM cells by RT-PCR and Immunohistochemistry assay. Three CDV strains were isolated successfully in Vero-rSLAM cells 36 -48 hours after inoculation with spleen or lung specimens from foxes and raccoon dogs with distemper. By contrast, no CDV was recovered from those CD positive specimens when Vero cells were used for virus isolation. Infected foxes and raccoon dogs with LN(10)f1 strain all showed typical CD symptoms and high mortality (2/3 for foxes and 3/3 for raccoon dogs) in 22 days post challenge. Our results indicate that Vero-rSLAM cells stably expressing raccoon dog SLAM are highly sensitive to CDV in clinical specimens and the CDV isolation can maintain high virulence to its host animals.

  17. Construction of recombinant ZNF230/GFP fused plasmids and their expression and cellular localization

    DEFF Research Database (Denmark)

    Xu, Wen-Ming; Zhang, Si-Zhong; Qiu, Wei-Min

    2004-01-01

    To use green fluorescent protein as a marker to study the localization of the fusion protein, the mutant full length cDNAs of human ZNF230 and mouse znf230 with their stop codon TGA changed to TGG were obtained by PCR amplification, and then cloned into pGEM-Teasy vector. After the double enzyme...... cutting, the mutated human and mouse ZNF230(znf230) were inserted into mammalian expression plasmid pEGFP-N1. Thus we constructed the plasmid with fusion gene of ZNF230 and green fluorescent protein(GFP). Then the Cos cell was transfected with the fused gene by liposome. Fluorescence microscopy showed...

  18. Construction and Development of a Cardiac Tissue-Specific and Hypoxia-Inducible Expression Vector

    Directory of Open Access Journals (Sweden)

    Shahrooz Ghaderi

    2018-03-01

    Full Text Available Purpose: Cardiovascular gene therapy is a sophisticated approach, thanks to the safety of vectors, stable transgene expression, delivery method, and different layers of the heart. To date, numerous expression vectors have been introduced in biotechnology and biopharmacy industries in relation to genetic manipulation. Despite the rapid growth of these modalities, they must be intelligently designed, addressing the cardiac-specific transgene expression and less side effects. Herein, we conducted a pilot project aiming to design a cardiac-specific hypoxia-inducible expression cassette. Methods: We explored a new approach to design an expression cassette containing cardiac specific enhancer, hypoxia response elements (HRE, cardiac specific promoter, internal ribosome entry site (IRES, and beta globin poly A sequence to elicit specific and inducible expression of the gene of interest. Enhanced green fluorescent protein (eGFP was sub-cloned by BglII and NotI into the cassette. The specificity and inducible expression of the cassette was determined in both mouse myoblast C2C12 and mammary glandular tumor 4T1 as ‘twin’ cells. eGFP expression was evaluated by immunofluorescence microscope and flow cytometry at 520 nm emission peak. Results: Our data revealed that the designed expression cassette provided tissue specific and hypoxia inducible (O2<1% transgene expression. Conclusion: It is suggested that cardiac-specific enhancer combined with cardiac-specific promoter are efficient for myoblast specific gene expression. As well, this is for the first time that HRE are derived from three well known hypoxia-regulated promoters. Therefore, there is no longer need to overlap PCR process for one repeated sequence just in one promoter.

  19. UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

    Directory of Open Access Journals (Sweden)

    Emanuela Chiarella

    Full Text Available Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6 where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in

  20. UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

    Science.gov (United States)

    Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

    2014-01-01

    Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and

  1. Identification, functional characterization and expression pattern of myeloid differentiation factor 88 (MyD88) in Sepiella japonica.

    Science.gov (United States)

    Huo, Liping; Bao, Miaomiao; Lv, Zhenming; Chi, Changfeng; Wang, Tianming; Liu, Huihui

    2018-05-01

    Myeloid differentiation factor 88 (MyD88) is an adaptor protein involved in the interleukin-1 receptor and Toll-like receptor-induced activation of nuclear factor-κB (NF-κB). In this study a novel isoform of MyD88 in Sepiella japonica (SjMyD88) was cloned and functionally characterized (GenBank accession no. AQY56781.1). The complete cDNA sequence of SjMyD88 was 1912 bp and contained a 1017 bp open reading frame encoding 338 amino acid residues, which was similar to its mollusk orthologues in the length. BLASTp analysis suggested the deduced amino acids sequence of SjMyD88 shared high identity to the known MyD88, for instance, 64% identity with Octopus bimaculoides. Sequence analysis revealed two conserved domains, the N-terminal DD and the C-terminal TIR domain appeared in SjMyD88, which was consistent with MyD88 proteins from other species. The fusion expression of SjMyD88 and green fluorescent protein (EGFP) in HEK293 cells was conducted and cytoplasm localization was detected. Meanwhile, the TIR-pmCherry fusion protein showed red fluorescence and mainly distributed in the cytoplasm. After cotransfection MyD88-EGFP and TIR-pmCherry red obviously overlapped and changed to yellowish green. The results suggested that there was the interaction between homologous TIR-pmcherry and MyD88-EGFP. Tissues expression profiles analysis showed that SjMyD88 ubiquitously expressed in all tested tissues with the highest expression in the gills and livers except reproductive related tissue, and it was significantly induced in livers under LPS stress. These data provide insight into the roles of SjMyD88 in the TLR signaling pathway of S. japonica in response to pathogenic bacteria. Copyright © 2018. Published by Elsevier Ltd.

  2. Manipulation of gene expression by infrared laser heat shock and its application to the study of tracheal development in Drosophila.

    Science.gov (United States)

    Miao, Guangxia; Hayashi, Shigeo

    2015-03-01

    Induction of gene expression in a specific cell and a defined time window is desirable to investigate gene function at the cellular level during morphogenesis. To achieve this, we attempted to introduce the infrared laser-evoked gene operator system (IR-LEGO, Kamei et al., 2009) in the Drosophila embryo. In this technique, infrared laser light illumination induces genes to be expressed under the control of heat shock promoters at the single cell level. We applied IR-LEGO to a transgenic fly stock, HS-eGFP, in which the enhanced green fluorescent protein (eGFP) gene is placed under the control of heat shock protein 70 promoter, and showed that eGFP expression can be induced in single cells within 1-2 hr after IR illumination. Furthermore, induction of HS-Branchless transgene encoding the Drosophila fibroblast growth factor (FGF) effectively altered the migration and branching patterns of the tracheal system. Our results indicated that IR-LEGO is a promising choice for the timely control of gene expression in a small group of cells in the Drosophila embryo. By using IR-LEGO, we further demonstrated that the tracheal terminal branching program is sensitive to localized expression of exogenous FGF. © 2014 Wiley Periodicals, Inc.

  3. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    International Nuclear Information System (INIS)

    Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-01

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

  4. Capillary arterialization requires the bone-marrow-derived cell (BMC)-specific expression of chemokine (C-C motif) receptor-2, but BMCs do not transdifferentiate into microvascular smooth muscle.

    Science.gov (United States)

    Nickerson, Meghan M; Burke, Caitlin W; Meisner, Joshua K; Shuptrine, Casey W; Song, Ji; Price, Richard J

    2009-01-01

    Chemokine (C-C motif) receptor-2 (CCR2) regulates arteriogenesis and angiogenesis, facilitating the MCP-1-dependent recruitment of growth factor-secreting bone marrow-derived cells (BMCs). Here, we tested the hypothesis that the BMC-specific expression of CCR2 is also required for new arteriole formation via capillary arterialization. Following non-ischemic saphenous artery occlusion, we measured the following in gracilis muscles: monocyte chemotactic protein-1 (MCP-1) in wild-type (WT) C57Bl/6J mice by ELISA, and capillary arterialization in WT-WT and CCR2(-/-)-WT (donor-host) bone marrow chimeric mice, as well as BMC transdifferentiation in EGFP(+)-WT mice, by smooth muscle (SM) alpha-actin immunochemistry. MCP-1 levels were significantly elevated 1 day after occlusion in WT mice. In WT-WT mice at day 7, compared to sham controls, arterial occlusion induced a 34% increase in arteriole length density, a 46% increase in SM alpha-actin(+) vessels, and a 45% increase in the fraction of vessels coated with SM alpha-actin, indicating significant capillary arterialization. However, in CCR2(-/-)-WT mice, no differences were observed between arterial occlusion and sham surgery. In EGFP(+)-WT mice, EGFP and SM alpha-actin never colocalized. We conclude that BMC-specific CCR2 expression is required for skeletal muscle capillary arterialization following arterial occlusion; however, BMCs do not transdifferentiate into smooth muscle.

  5. Visual expression analysis of the responses of the alternative oxidase gene (aox1) to heat shock, oxidative, and osmotic stresses in conidia of citric acid-producing Aspergillus niger.

    Science.gov (United States)

    Honda, Yuki; Hattori, Takasumi; Kirimura, Kohtaro

    2012-03-01

    The citric acid-producing filamentous fungus Aspergillus niger WU-2223L shows cyanide-insensitive respiration catalyzed by alternative oxidase in addition to the cytochrome pathway. Sequence analysis of the 5' flanking region of the alternative oxidase gene (aox1) revealed a potential heat shock element (HSE) and a stress response element (STRE). We have previously confirmed aox1 expression in conidia. In this study, to confirm whether the upstream region of aox1 responds to various stresses, we used a visual expression analysis system for single-cell conidia of the A. niger strain AOXEGFP-1. This strain harbored a fusion gene comprising aox1 and egfp, which encodes the enhanced green fluorescent protein (EGFP). The fluorescence intensity of EGFP increased in conidia of A. niger AOXEGFP-1 that were subjected to heat shock at 35-45 °C, oxidative stress by exposure to 5mM paraquat or 1 mM t-butylhydroperoxide, or osmotic stresses by exposure to 0.5 M KCl or 1.0 M mannitol. These results indicate that the putative HSE and STRE in the upstream region of aox1 directly or indirectly respond to heat shock, oxidative, and osmotic stresses. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Molecular cloning and tissue-specific expression analysis of mouse spinesin, a type II transmembrane serine protease 5

    International Nuclear Information System (INIS)

    Watanabe, Yoshihisa; Okui, Akira; Mitsui, Shinichi; Kawarabuki, Kentaro; Yamaguchi, Tatsuyuki; Uemura, Hidetoshi; Yamaguchi, Nozomi

    2004-01-01

    We have previously reported novel serine proteases isolated from cDNA libraries of the human and mouse central nervous system (CNS) by PCR using degenerate oligodeoxyribonucleotide primers designed on the basis of the serine protease motifs, AAHC and DSGGP. Here we report a newly isolated serine protease from the mouse CNS. This protease is homologous (77.9% identical) to human spinesin type II transmembrane serine protease 5. Mouse spinesin (m-spinesin) is also composed of (from the N-terminus) a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger-receptor-like domain, and a serine protease domain, as is h-spinesin. We also isolated type 1, type 2, and type 3 variant cDNAs of m-spinesin. Full-length spinesin (type 4) and type 3 contain all the domains, whereas type 1 and type 2 variants lack the cytoplasmic, transmembrane, and scavenger-receptor-like domains. Subcellular localization of the variant forms was analyzed using enhanced green fluorescent protein (EGFP) fusion proteins. EGFP-type 4 fusion protein was predominantly localized to the ER, Golgi apparatus, and plasma membrane, whereas EGFP-type 1 was localized to the cytoplasm, reflecting differential classification of m-spinesin variants into transmembrane and cytoplasmic types. We analyzed the distribution of m-spinesin variants in mouse tissues, using RT-PCR with variant-specific primer sets. Interestingly, transmembrane-type spinesin, types 3 and 4, was specifically expressed in the spinal cord, whereas cytoplasmic type, type 1, was expressed in multiple tissues, including the cerebrum and cerebellum. Therefore, m-spinesin variants may have distinct biological functions arising from organ-specific variant expression

  7. Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

    Science.gov (United States)

    Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

    2009-12-30

    Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements

  8. Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

    Science.gov (United States)

    2009-01-01

    Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T

  9. ФC31 Integrase-Mediated Isolation and Characterization of Novel Safe Harbors for Transgene Expression in the Pig Genome

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    Bi, Yanzhen; Hua, Zaidong; Ren, Hongyan; Zhang, Liping; Xiao, Hongwei; Liu, Ximei; Hua, Wenjun; Mei, Shuqi; Molenaar, Adrian; Laible, Götz; Zheng, Xinmin

    2018-01-01

    Programmable nucleases have allowed the rapid development of gene editing and transgenics, but the technology still suffers from the lack of predefined genetic loci for reliable transgene expression and maintenance. To address this issue, we used ФC31 integrase to navigate the porcine genome and identify the pseudo attP sites suitable as safe harbors for sustained transgene expression. The combined ФC31 integrase mRNA and an enhanced green fluorescence protein (EGFP) reporter donor were microinjected into one-cell zygotes for transgene integration. Among the resulting seven EGFP-positive piglets, two had transgene integrations at pseudo attP sites, located in an intergenic region of chromosome 1 (chr1-attP) and the 6th intron of the TRABD2A gene on chromosome 3 (chr3-attP), respectively. The integration structure was determined by TAIL-PCR and Southern blotting. Primary fibroblast cells were isolated from the two piglets and examined using fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA), which demonstrated that the chr1-attP site was more potent than chr3-attP site in supporting the EGFP expression. Both piglets had green feet under the emission of UV light, and pelleted primary fibroblast cells were green-colored under natural light, corroborating that the two pseudo attP sites are beneficial to transgene expression. The discovery of these two novel safe harbors for robust and durable transgene expression will greatly facilitate the use of transgenic pigs for basic, biomedical and agricultural studies and applications. PMID:29300364

  10. Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense

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    Yuki Horiuchi

    2018-01-01

    Full Text Available Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense. We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein “ember” from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 105 M−1·cm−1. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

  11. A construct with fluorescent indicators for conditional expression of miRNA

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    Xia Xugang

    2008-10-01

    Full Text Available Abstract Background Transgenic RNAi holds promise as a simple, low-cost, and fast method for reverse genetics in mammals. It may be particularly useful for producing animal models for hypomorphic gene function. Inducible RNAi that permits spatially and temporally controllable gene silencing in vivo will enhance the power of transgenic RNAi approach. Furthermore, because microRNA (miRNA targeting specific genes can be expressed simultaneously with protein coding genes, incorporation of fluorescent marker proteins can simplify the screening and analysis of transgenic RNAi animals. Results We sought to optimally express a miRNA simultaneously with a fluorescent marker. We compared two construct designs. One expressed a red fluorescent protein (RFP and a miRNA placed in its 3' untranslated region (UTR. The other expressed the same RFP and miRNA, but the precursor miRNA (pre-miRNA coding sequence was placed in an intron that was inserted into the 3'-UTR. We found that the two constructs expressed comparable levels of miRNA. However, the intron-containing construct expressed a significantly higher level of RFP than the intron-less construct. Further experiments indicate that the 3'-UTR intron enhances RFP expression by its intrinsic gene-expression-enhancing activity and by eliminating the inhibitory effect of the pre-miRNA on the expression of RFP. Based on these findings, we incorporated the intron-embedded pre-miRNA design into a conditional expression construct that employed the Cre-loxP system. This construct initially expressed EGFP gene, which was flanked by loxP sites. After exposure to Cre recombinase, the transgene stopped EGFP expression and began expression of RFP and a miRNA, which silenced the expression of specific cellular genes. Conclusion We have designed and tested a conditional miRNA-expression construct and showed that this construct expresses both the marker genes strongly and can silence the target gene efficiently upon Cre

  12. Actin Nemaline Myopathy Mouse Reproduces Disease, Suggests Other Actin Disease Phenotypes and Provides Cautionary Note on Muscle Transgene Expression

    Science.gov (United States)

    Ravenscroft, Gianina; Jackaman, Connie; Sewry, Caroline A.; McNamara, Elyshia; Squire, Sarah E.; Potter, Allyson C.; Papadimitriou, John; Griffiths, Lisa M.; Bakker, Anthony J.; Davies, Kay E.; Laing, Nigel G.; Nowak, Kristen J.

    2011-01-01

    Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ∼30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations. PMID:22174871

  13. Establishment and Identification of Chinese Hamster Ovary Cell Lines with Stable Expression of Soluble CD40 Ligands

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    JIANG Hua-wei

    2014-09-01

    Full Text Available Objective: To establish the Chinese Hamster Ovary (CHO cell lines with stable expression of soluble CD40 ligands (sCD40L. Methods: Recombinant plasmid pIRES2-EGFP-sCD40L, enzyme digestion and sequencing identification were obtained by cloning sCD40L coding sequences into eukaryotic expression vector pIRES2-EGFP from carrier pDC316-sCD40 containing sCD40L. CHO cells were transfected by electroporation, followed by screening of resistant clones with G418, after which monoclones were obtained by limited dilution assay and multiply cultured. Flow cytometer and reverted fluorescence microscope were applied to observe the expression of green fluorescent protein, while sCD40L expression was detected by polymerase chain reaction (PCR, reverse transcription-polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA from aspects of deoxyribose nucleic acid (DNA, messenger ribonucleic acid (mRNA and protein, respectively. CHO-sCD40L was cultured together with MDA-MB-231 cells to compare the expression changes of surface molecule fatty acid synthase (Fas by flow cytometer and observe the apoptosis of MDA-MB-231 cells after Fas activated antibodies (CH-11 were added 24 h later. Results: Plasmid pIRES2-EGFP-sCD40L was successfully established, and cell lines with stable expression of sCD40L were obtained with cloned culture after CHO cell transfection, which was named as B11. Flow cytometer and reverted fluorescence microscope showed >90% expression of green fluorescent protein, while PCR, RT-PCR and ELISA suggested integration of sCD40L genes into cell genome DNA, transcription of sCD40L mRNA and sCD40L protein expression being (4.5±2.1 ng/mL in the supernatant of cell culture, respectively. After co-culture of B11 and MDA-MB-231 cells, the surface Fas expression of MDA-MB-231 cells was increased from (3±1.02 % to (34.8±8.75%, while the apoptosis rate 24 h after addition of CH11 from (5.4±1.32% to (20.7±5.24%, and the differences

  14. Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI

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    Margison Geoffrey P

    2006-03-01

    Full Text Available Abstract Background A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES sequence of encephalomyocarditis virus (EMCV and/or foot-and-mouth disease virus (FMDV cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP, O6-methylguanine-DNA-methyltransferase (MGMT, and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. Results All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector. Conclusion The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.

  15. Efficient production of antibody Fab fragment by transient gene expression in insect cells.

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    Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

    2017-08-01

    Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. Social Isolation Modulates CLOCK Protein and Beta-Catenin Expression Pattern in Gonadotropin-Inhibitory Hormone Neurons in Male Rats

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    Chuin Hau Teo

    2017-09-01

    Full Text Available Postweaning social isolation reduces the amplitude of the daily variation of CLOCK protein in the brain and induces lower reproductive activity. Gonadotropin-inhibitory hormone (GnIH acts as an inhibitor in the reproductive system and has been linked to stress. Social isolation has been shown to lower neuronal activity of GnIH-expressing neurons in the dorsomedial hypothalamus (DMH. The exact mechanism by which social isolation may affect GnIH is still unclear. We investigated the impact of social isolation on regulatory cellular mechanisms in GnIH neurons. We examined via immunohistochemistry the expression of CLOCK protein at four different times throughout the day in GnIH cells tagged with enhanced fluorescent green protein (EGFP-GnIH in 9-week-old adult male rats that have been raised for 6 weeks under postweaning social isolation and compared them with group-raised control rats of the same age. We also studied the expression of β-catenin—which has been shown to be affected by circadian proteins such as Bmal1—in EGFP-GnIH neurons to determine whether it could play a role in linking CLOCK in GnIH neurons. We found that social isolation modifies the pattern of CLOCK expression in GnIH neurons in the DMH. Socially isolated rats displayed greater CLOCK expression in the dark phase, while control rats displayed increased CLOCK expression in the light phase. Furthermore, β-catenin expression pattern in GnIH cells was disrupted by social isolation. This suggests that social isolation triggers changes in CLOCK and GnIH expression, which may be associated with an increase in nuclear β-catenin during the dark phase.

  17. Social Isolation Modulates CLOCK Protein and Beta-Catenin Expression Pattern in Gonadotropin-Inhibitory Hormone Neurons in Male Rats.

    Science.gov (United States)

    Teo, Chuin Hau; Soga, Tomoko; Parhar, Ishwar S

    2017-01-01

    Postweaning social isolation reduces the amplitude of the daily variation of CLOCK protein in the brain and induces lower reproductive activity. Gonadotropin-inhibitory hormone (GnIH) acts as an inhibitor in the reproductive system and has been linked to stress. Social isolation has been shown to lower neuronal activity of GnIH-expressing neurons in the dorsomedial hypothalamus (DMH). The exact mechanism by which social isolation may affect GnIH is still unclear. We investigated the impact of social isolation on regulatory cellular mechanisms in GnIH neurons. We examined via immunohistochemistry the expression of CLOCK protein at four different times throughout the day in GnIH cells tagged with enhanced fluorescent green protein (EGFP-GnIH) in 9-week-old adult male rats that have been raised for 6 weeks under postweaning social isolation and compared them with group-raised control rats of the same age. We also studied the expression of β-catenin-which has been shown to be affected by circadian proteins such as Bmal1-in EGFP-GnIH neurons to determine whether it could play a role in linking CLOCK in GnIH neurons. We found that social isolation modifies the pattern of CLOCK expression in GnIH neurons in the DMH. Socially isolated rats displayed greater CLOCK expression in the dark phase, while control rats displayed increased CLOCK expression in the light phase. Furthermore, β-catenin expression pattern in GnIH cells was disrupted by social isolation. This suggests that social isolation triggers changes in CLOCK and GnIH expression, which may be associated with an increase in nuclear β-catenin during the dark phase.

  18. Relationship between expression level of hygromycin B-resistant gene and Agrobacterium tumefaciens-mediated transformation efficiency in Beauveria bassiana JEF-007.

    Science.gov (United States)

    Nai, Y S; Lee, M R; Kim, S; Lee, S J; Kim, J C; Yang, Y T; Kim, J S

    2017-09-01

    Agrobacterium tumefaciens-mediated transformation (AtMT) is an effective method for generation of entomopathogenic Beauveria bassiana transformants. However, some strains grow on the selective medium containing hygromycin B (HygB), which reduces the selection efficiency of the putative transformants. In this work, a relationship between HygB resistance gene promoter and AtMT efficiency was investigated to improve the transformant selection. Ten B. bassiana isolates were grown on 800 μg ml -1 HygB medium, but only JEF-006, -007 and -013 showed susceptibility to the antibiotics. Particularly, JEF-007 showed the most dose-dependent susceptibility. Two different Ti-Plasmids, pCeg (gpdA promoter based) and pCambia-egfp (CaMV 35S promoter based), were constructed to evaluate the promoters on the expression of HygB resistance gene (hph) at 100, 150 and 200 μg ml -1 HygB medium. Eight days after the transformation, wild type, AtMT/pCeg and AtMT/pCambia-egfp colonies were observed on 100 μg ml -1 HygB, but significantly larger numbers of colonies were counted on AtMT/pCeg plates. At higher HygB concentration (150 μg ml -1 ), only AtMT/pCeg colonies were further observed, but very few colonies were observed on the wild type and AtMT/pCambia-egfp plates. Putative transformants were subjected to PCR, RT-PCR and qRT-PCR to investigate the T-DNA insertion rate and gene expression level. Consequently, >80% of colonies showed successful AtMT transformation, and the hph expression level in AtMT/pCeg colonies was higher than that of AtMT/pCambia-egfp colonies. In the HygB-susceptible B. bassianaJEF-007, gpdA promoter works better than CaMV 35S promoter in the expression of HygB resistance gene at 150 μg ml -1 HygB, consequently improving the selection efficiency of putative transformants. These results provide useful information for determining AtMT effectiveness in B. bassiana isolates, particularly antibiotic susceptibility and the role of promoters. © 2017 The

  19. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

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    Noireaux Vincent

    2010-06-01

    Full Text Available Abstract Background Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. Results In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP regeneration systems: creatine phosphate (CP, phosphoenolpyruvate (PEP, and 3-phosphoglyceric acid (3-PGA. The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. Conclusions Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma

  20. ­Glial and stem cell expression of murine Fibroblast Growth Factor Receptor 1 in the embryonic and perinatal nervous system

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    Jantzen C. Collette

    2017-06-01

    Full Text Available Background Fibroblast growth factors (FGFs and their receptors (FGFRs are involved in the development and function of multiple organs and organ systems, including the central nervous system (CNS. FGF signaling via FGFR1, one of the three FGFRs expressed in the CNS, stimulates proliferation of stem cells during prenatal and postnatal neurogenesis and participates in regulating cell-type ratios in many developing regions of the brain. Anomalies in FGFR1 signaling have been implicated in certain neuropsychiatric disorders. Fgfr1 expression has been shown, via in situ hybridization, to vary spatially and temporally throughout embryonic and postnatal development of the brain. However, in situ hybridization lacks sufficient resolution to identify which cell-types directly participate in FGF signaling. Furthermore, because antibodies raised against FGFR1 commonly cross-react with other members of the FGFR family, immunocytochemistry is not alone sufficient to accurately document Fgfr1 expression. Here, we elucidate the identity of Fgfr1 expressing cells in both the embryonic and perinatal mouse brain. Methods To do this, we utilized a tgFGFR1-EGFPGP338Gsat BAC line (tgFgfr1-EGFP+ obtained from the GENSAT project. The tgFgfr1-EGFP+ line expresses EGFP under the control of a Fgfr1 promoter, thereby causing cells endogenously expressing Fgfr1 to also present a positive GFP signal. Through simple immunostaining using GFP antibodies and cell-type specific antibodies, we were able to accurately determine the cell-type of Fgfr1 expressing cells. Results This technique revealed Fgfr1 expression in proliferative zones containing BLBP+ radial glial stem cells, such as the cortical and hippocampal ventricular zones, and cerebellar anlage of E14.5 mice, in addition to DCX+ neuroblasts. Furthermore, our data reveal Fgfr1 expression in proliferative zones containing BLBP+ cells of the anterior midline, hippocampus, cortex, hypothalamus, and cerebellum of P0.5 mice

  1. Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

    Science.gov (United States)

    Sharifi Tabar, Mehdi; Hesaraki, Mahdi; Esfandiari, Fereshteh; Sahraneshin Samani, Fazel; Vakilian, Haghighat; Baharvand, Hossein

    2015-01-01

    Objective Genetic modification of human embryonic stem cells (hESCs) is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest (GOI) into the target cell line, however, there are few re- ports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor. Materials and Methods In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for tran- sient expression of green fluorescent protein in two genetically different hESC lines, Roy- an H5 (XX) and Royan H6 (XY), as well as human foreskin fibroblasts (hFF). For long-term EGFP expression VASA and OLIG2 promoters (germ cell and motoneuron specific genes, respectively), were isolated and subsequently cloned into a pBluMAR5 plasmid backbone to drive EGFP expression. Flow cytometry analysis was performed two days after trans- fection to determine transient expression efficiency. Differentiation of drug resistant hESC colonies toward primordial germ cells (PGCs) was conducted to confirm stable integration of the transgene. Results Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation ef- ficiency revealed that the vector concentrations from 20-60 μg and the density of the sub- jected cells (5×105and 1×106cells) were not as effective as the genetic background and voltage rate. The present data indicated that in contrast to the circular form, the linearized vector generated more distinctive drug resistant colonies. Conclusion

  2. [Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system].

    Science.gov (United States)

    Lu, Yue; Liu, Dan; Zhang, Xiaoren; Liu, Xuerong; Shen, Wei; Zheng, Gang; Liu, Yunfan; Dong, Xiaoyan; Wu, Xiaobing; Gao, Jimin

    2011-08-01

    We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

  3. Comparing Effects of BK Virus Agnoprotein and Herpes Simplex-1 ICP47 on MHC-I and MHC-II Expression

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    Michela Cioni

    2013-01-01

    Full Text Available Background. Among human polyomaviruses, only BK virus (BKV and JC virus (JCV encode an agnoprotein upstream of VP1 on the viral late transcript. BKV agnoprotein is abundantly expressed late in the viral life cycle, but specific cellular and humoral immune responses are low or absent. We hypothesized that agnoprotein might contribute to BKV immune evasion by downregulating HLA expression, similar to Herpes simplex virus-1 ICP47. Methods UTA-6 or primary human renal proximal tubular epithelial cells (RPTEC were co-transfected with plasmids constitutively expressing agnoprotein, or ICP47, and enhanced green-fluorescent protein (EGFP. EGFP-gated cells were analyzed for HLA-ABC and HLA-DR expression by flow cytometry. HLA-ABC and HLA-DR expression was also analyzed on UTA-6 bearing tetracycline-regulated agnoprotein or ICP47. Effects of agnoprotein on viral peptide-dependent T-cell killing were investigated using 51Cr release. Results. ICP47 downregulated HLA-ABC without affecting HLA-DR, whereas agnoprotein did not affect HLA-ABC or HLA-DR expression. Interferon-γ treatment increased HLA-ABC in a dose-dependent manner, which was antagonized by ICP47, but not by agnoprotein. In UTA-6 cells, agnoprotein expression did neither impair HLA-ABC or -DR expression nor peptide-specific killing impaired by HLA-matched T-cells. Conclusion. Unlike the HSV-1 ICP47, BKV agnoprotein does not contribute to viral immune evasion by down-regulating HLA-ABC, or interfere with HLA-DR expression or peptide-dependent T-cell cytotoxicity.

  4. Expression of transgenes targeted to the Gt(ROSA26Sor locus is orientation dependent.

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    Douglas Strathdee

    2006-12-01

    Full Text Available Targeting transgenes to a chosen location in the genome has a number of advantages. A single copy of the DNA construct can be inserted by targeting into regions of chromatin that allow the desired developmental and tissue-specific expression of the transgene.In order to develop a reliable system for reproducibly expressing transgenes it was decided to insert constructs at the Gt(ROSA26Sor locus. A cytomegalovirus (CMV promoter was used to drive expression of the Tetracycline (tet transcriptional activator, rtTA2(s-M2, and test the effectiveness of using the ROSA26 locus to allow transgene expression. The tet operator construct was inserted into one allele of ROSA26 and a tet responder construct controlling expression of EGFP was inserted into the other allele.Expression of the targeted transgenes was shown to be affected by both the presence of selectable marker cassettes and by the orientation of the transgenes with respect to the endogenous ROSA26 promoter. These results suggest that transcriptional interference from the endogenous gene promoter or from promoters in the selectable marker cassettes may be affecting transgene expression at the locus. Additionally we have been able to determine the optimal orientation for transgene expression at the ROSA26 locus.

  5. Dual-color fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells.

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    Saori Tsuji

    Full Text Available Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps.

  6. Nerve growth factor expression by PLG-mediated lipofection.

    Science.gov (United States)

    Whittlesey, Kevin J; Shea, Lonnie D

    2006-04-01

    Biomaterials capable of efficient gene delivery provide a fundamental tool for basic and applied research models, such as promoting neural regeneration. We developed a system for the encapsulation and sustained release of plasmid DNA complexed with a cationic lipid and investigated their efficacy using in vitro models of neurite outgrowth. Sustained lipoplex release was obtained for up to 50 days, with rates controlled by the fabrication conditions. Released lipoplexes retained their activity, transfecting 48.2+/-8.3% of NIH3T3 cells with luciferase activity of 3.97x10(7)RLU/mg. Expression of nerve growth factor (NGF) was employed in two models of neurite outgrowth: PC12 and primary dorsal root ganglia (DRG) co-culture. Polymer-mediated lipofection of PC12 produced bioactive NGF, eliciting robust neurite outgrowth. An EGFP/NGF dual-expression vector identified transfected cells (GFP-positive) while neurite outgrowth verified NGF secretion. A co-culture model examined the ability of NGF secretion by an accessory cell population to stimulate DRG neurite outgrowth. Polymer-mediated transfection of HEK293T with an NGF-encoding plasmid induced outgrowth by DRG neurons. This system could be fabricated as implants or nerve guidance conduits to support cellular and tissue regeneration. Combining this physical support with the ability to locally express neurotrophic factors will potentiate regeneration in nerve injury and disease models.

  7. High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

    Science.gov (United States)

    Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

    2016-03-01

    Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (~7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Establishment of A Malignant Pleural Effusion Mouse Model with Lewis Lung 
Carcinoma Cell Lines Expressing Enhanced Green Fluorescent Protein

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    Xingqun MA

    2012-06-01

    Full Text Available Background and objective Malignant pleural effusion (MPE is a poor prognosis factor in patients with advanced lung cancer. The aim of this study is to establish a mouse model of MPE using Lewis lung carcinoma (LLC cell lines expressing enhanced green fluorescent protein (EGFP. Methods The mouse model was created by injecting LLC-EGFP cells directly into the pleural cavity of mice that were sacrificed periodically. The dynamic growth and metastasis of tumor cells were screened using in vivo fluorescence imaging. The remaining mice were subjected to transverse computed tomography (CT imaging periodically to analyze the formation rate of pleural effusion. The survival rate and tumor metastasis were also observed. Pleural fluid was gently aspirated using a 1 mL syringe and its volume was measured. When two or more mice bore pleural effusion at the same time, we calculated the average volume. The correlation of pleural effusion with the integrated optical density (IOD were analyzed. Results Four days after the inoculation of LLC-EGFP cells, green fluorescence was observed by opening the chest wall. The tumor formation rate was 100%, and the IOD gradually increased after inoculation. The metastasis sites were mediastinal, and the hilar lymph nodes were contralateral pleural as well as pericardial. The metastasis rates were 87%, 73% and 20%, respectively. The CT scan revealed that the formation rates of pleural effusion on days 7, 14 and 21 were 13%, 46% and 53%, respectively. The average volume of pleural effusion increased obviously on day 10 and peaked on day 16 with a value of 0.5 mL. The mean survival time of nude mice was 28.8 days. The volume of pleural effusion and IOD were significantly correlated (r=0.91, P<0.000,1. Conclusion A mouse model of lung cancer malignant pleural effusion was successfully established by injecting LLC lines expressing EGFP into the pleural cavity under a microscope. The model can enable dynamic observations of the

  9. Path Expressions

    Science.gov (United States)

    1975-06-01

    Traditionally, synchronization of concurrent processes is coded in line by operations on semaphores or similar objects. Path expressions move the...discussion about a variety of synchronization primitives . An analysis of their relative power is found in [3]. Path expressions do not introduce yet...another synchronization primitive . A path expression relates to such primitives as a for- or while-statement of an ALGOL-like language relates to a JUMP

  10. [Gene transfer-induced human heme oxygenase-1 over-expression protects kidney from ischemia-reperfusion injury in rats].

    Science.gov (United States)

    Lü, Jin-xing; Yan, Chun-yin; Pu, Jin-xian; Hou, Jian-quan; Yuan, He-xing; Ping, Ji-gen

    2010-12-14

    To study the protection of gene transfer-induced human heme oxygenase-1 over-expression against renal ischemia reperfusion injury in rats. The model of kidney ischemia-reperfusion injury was established with Sprague-Dawley rats. In the therapy group (n=18), the left kidney was perfused and preserved with Ad-hHO-1 at 2.5×10(9) pfu/1.0 ml after flushed with 0-4°C HC-A organ storage solution via donor renal aorta. The rats in control groups were perfused with 0.9% saline solution (n=12) or the vector carrying no interest gene Ad-EGFP 2.5×10(9) pfu/1.0 ml (n=18) instead of Ad-hHO-1. BUN and Cr in serum were measured by slide chemical methods. The kidney samples of rats were harvested for assay of histology, immunohistochemistry and quantification of HO enzymatic activity. Apoptosis cells in the kidney were measured by TUNEL. Ad-hHO-1 via donor renal aorta could transfect renal cells of rats effectively, enzymatic activity of HO in treated group [(1.62±0.07) nmol×mg(-1)×min(-1)] is higher than in control groups treated with saline solution team [(1.27±0.07) nmol×mg(-1)×min(-1)] and vector EGFP team [(1.22±0.06) nmol×mg(-1)×min(-1)] (PhHO-1 expressed hHO-1 in kidneys at a high level. Corresponding to this, the level of BUN and Cr, as well as the number of apoptosis cells, were decreased, and the damage in histology by HE staining was ameliorated. Over-expression of human HO-1 can protect the kidney from ischemia/reperfusion injury in rats.

  11. Clone and expression of human transferrin receptor gene: a marker gene for magnetic resonance imaging

    International Nuclear Information System (INIS)

    Li Li; Liu Lizhi; Lv Yanchun; Liu Xuewen; Cui Chunyan; Wu Peihong; Liu Qicai; Ou Shanxing

    2007-01-01

    Objective: To clone human transferrin receptor (hTfR) gene and construct expression vector producing recombination protein. Methods: Human transferrin receptor gene cDNA was amplified by RT-PCR from human embryonic liver and lung tissue. Recombinant pcDNA3-hTfR and pEGFP-Cl-hTfR plasmids were constructed and confirmed by DNA sequencing. These plasmids were stably transfected into the HEK293 cells. The protein expression in vitro was confirmed by Western Blot. The efficiency of expression and the location of hTfR were also investigated by fluorescence microscopy and confocal fluorescence microscopy. Results: The full length cDNA of hTfR gene (2332 bp) was cloned and sequenced. The hTfR (190 000) was overexpressed in transfected HEK293 cells by Western blot analysis. Fluorescence micrographs displayed that the hTfR was expressed at high level and located predominantly in the cell surface. Conclusions: Human transferrin receptor (hTfR) gene has been successfully cloned and obtained high-level expression in HEK293 cells, and the recombination protein of hTfR distributed predominantly in the cell membrane. (authors)

  12. 'Fluorescent Cell Chip' for immunotoxicity testing: Development of the c-fos expression reporter cell lines

    International Nuclear Information System (INIS)

    Trzaska, Dominika; Zembek, Patrycja; Olszewski, Maciej; Adamczewska, Violetta; Ulleras, Erik; Dastych, JarosIaw

    2005-01-01

    The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals

  13. Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation

    International Nuclear Information System (INIS)

    Moen, Ingrid; Øyan, Anne M; Stuhr, Linda EB; Jevne, Charlotte; Wang, Jian; Kalland, Karl-Henning; Chekenya, Martha; Akslen, Lars A; Sleire, Linda; Enger, Per Ø; Reed, Rolf K

    2012-01-01

    The tumor microenvironment is pivotal in tumor progression. Thus, we aimed to develop a mammary tumor model to elucidate molecular characteristics in the stroma versus the tumor cell compartment by global gene expression. Secondly, since tumor hypoxia influences several aspects of tumor pathophysiology, we hypothesized that hyperoxia might have an inhibitory effect on tumor growth per se. Finally, we aimed to identify differences in gene expression and key molecular mechanisms, both in the native state and following treatment. 4T1 dsRed breast cancer cells were injected into eGFP expressing NOD/SCID mice. Group 1 was exposed to 3 intermittent HBO treatments (Day 1, 4 and 7), Group 2 to 7 daily HBO treatments (both 2.5bar, 100% O 2 , à 90 min), whereas the controls were exposed to a normal atmosphere. Tumor growth, histology, vascularisation, cell proliferation, cell death and metastasis were assessed. Fluorescence-activated cell sorting was used to separate tumor cells from stromal cells prior to gene expression analysis. The purity of sorted cells was verified by fluorescence microscopy. Gene expression profiling demonstrated that highly expressed genes in the untreated tumor stroma included constituents of the extracellular matrix and matrix metalloproteinases. Tumor growth was significantly inhibited by HBO, and the MAPK pathway was found to be significantly reduced. Immunohistochemistry indicated a significantly reduced microvessel density after intermittent HBO, whereas daily HBO did not show a similar effect. The anti-angiogenic response was reflected in the expression trends of angiogenic factors. The present in vivo mammary tumor model enabled us to separate tumor and stromal cells, and demonstrated that the two compartments are characterized by distinct gene expressions, both in the native state and following HBO treatments. Furthermore, hyperoxia induced a significant tumor growth-inhibitory effect, with significant down-regulation of the MAPK pathway

  14. Gene expression

    International Nuclear Information System (INIS)

    Hildebrand, C.E.; Crawford, B.D.; Walters, R.A.; Enger, M.D.

    1983-01-01

    We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn 2+ or Cd 2+ . We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

  15. Ectopic expression of AID in a non-B cell line triggers A:T and G:C point mutations in non-replicating episomal vectors.

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    Tihana Jovanic

    Full Text Available Somatic hypermutation (SHM of immunoglobulin genes is currently viewed as a two step process initiated by the deamination of deoxycytidine (C to deoxyuridine (U, catalysed by the activation induced deaminase (AID. Phase 1 mutations arise from DNA replication across the uracil residue or the abasic site, generated by the uracil-DNA glycosylase, yielding transitions or transversions at G:C pairs. Phase 2 mutations result from the recognition of the U:G mismatch by the Msh2/Msh6 complex (MutS Homologue, followed by the excision of the mismatched nucleotide and the repair, by the low fidelity DNA polymerase eta, of the gap generated by the exonuclease I. These mutations are mainly focused at A:T pairs. Whereas in activated B cells both G:C and A:T pairs are equally targeted, ectopic expression of AID was shown to trigger only G:C mutations on a stably integrated reporter gene. Here we show that when using non-replicative episomal vectors containing a GFP gene, inactivated by the introduction of stop codons at various positions, a high level of EGFP positive cells was obtained after transient expression in Jurkat cells constitutively expressing AID. We show that mutations at G:C and A:T pairs are produced. EGFP positive cells are obtained in the absence of vector replication demonstrating that the mutations are dependent only on the mismatch repair (MMR pathway. This implies that the generation of phase 1 mutations is not a prerequisite for the expression of phase 2 mutations.

  16. Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems.

    Science.gov (United States)

    Gagoski, Dejan; Mureev, Sergey; Giles, Nichole; Johnston, Wayne; Dahmer-Heath, Mareike; Škalamera, Dubravka; Gonda, Thomas J; Alexandrov, Kirill

    2015-02-10

    Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximize throughput and minimize the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1 ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in Leishmania tarentolae and Escherichia coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Ghrelin receptor expression and colocalization with anterior pituitary hormones using a GHSR-GFP mouse line.

    Science.gov (United States)

    Reichenbach, Alex; Steyn, Frederik J; Sleeman, Mark W; Andrews, Zane B

    2012-11-01

    Ghrelin is the endogenous ligand for the GH secretagogue receptor (GHSR) and robustly stimulates GH release from the anterior pituitary gland. Ghrelin also regulates the secretion of anterior pituitary hormones including TSH, LH, prolactin (PRL), and ACTH. However, the relative contribution of a direct action at the GHSR in the anterior pituitary gland vs. an indirect action at the GHSR in the hypothalamus remains undefined. We used a novel GHSR-enhanced green fluorescent protein (eGFP) reporter mouse to quantify GHSR coexpression with GH, TSH, LH, PRL, and ACTH anterior pituitary cells in males vs. females and in chow-fed or calorie-restricted (CR) mice. GHSR-eGFP-expressing cells were only observed in anterior pituitary. The number of GHSR-eGFP-expressing cells was higher in male compared with females, and CR did not affect the GHSR-eGFP cell number. Double staining revealed 77% of somatotrophs expressed GHSR-eGFP in both males and females. Nineteen percent and 12.6% of corticotrophs, 21% and 9% of lactotrophs, 18% and 19% of gonadotrophs, and 3% and 9% of males and females, respectively, expressed GHSR-eGFP. CR increased the number of TSH cells, but suppressed the number of lactotrophs and gonadotrophs, expressing GHSR-eGFP compared with controls. These studies support a robust stimulatory action of ghrelin via the GHSR on GH secretion and identify a previously unknown sexual dimorphism in the GHSR expression in the anterior pituitary. CR affects GHSR-eGFP expression on lactotrophs, gonadotrophs, and thyrotrophs, which may mediate reproductive function and energy metabolism during periods of negative energy balance. The low to moderate expression of GHSR-eGFP suggests that ghrelin plays a minor direct role on remaining anterior pituitary cells.

  18. Immunogenicity and Efficacy of Live L. tarentolae Expressing KMP11-NTGP96-GFP Fusion as a Vaccine Candidate against Experimental Visceral Leishmaniasis Caused by L. infantum

    Directory of Open Access Journals (Sweden)

    Vahid NASIRI

    2016-10-01

    Full Text Available Background: The aim of present study was to evaluate the protective efficacy of live recombinant L. tarentolae expressing KMP11-NTGP96-GFP fusion as candidates for live engineered recombinant vaccine against visceral leishmaniasis in BALB/c mice.Methods: KMP-11 and NT-GP96 genes cloned into the pJET1.2/blunt cloning vector and then into pEGFP-N1 expression vector. The KMP-11, NT-GP96 and GFP fused in pEGFP-N1 and subcloned into Leishmanian pLEXSY-neo vector. Finally this construct was transferred to L. tarentolae by electroporation. Tranfection was confirmed by SDS-PAGE, WESTERN blot, flowcytometry and RT-PCR. Protective efficacy of this construct was evaluated as a vaccine candidate against visceral leishmaniasis. Parasite burden, humoral and cellular immune responses were assessed before and at 4 weeks after challenge.Results: KMP- NT-Gp96-GFP Fusion was cloned successfully into pLEXSY -neo vector and this construct successfully transferred to L. tarentolae. Finding indicated that immunization with L. tarentolae tarentolae-KMP11-NTGP96-GFP provides significant protection against visceral leishmaniasis and was able to induce an increased expression of IFN-γ and IgG2a. Following challenge, a reduced parasite load in the spleen of the KMP11-NTGP96-GFP immunized group was detected.Conclusion: The present study is the first to use a combination of a Leishmania antigen with an immunologic antigen in live recombinant L. tarentolae and results suggest that L. tarentolae-KMP11-NTGP96-GFP could be considered as a potential tool in vaccination against visceral leishmaniasis and this vaccination strategy could provide a potent rout for future vaccine development. 

  19. Characterization, expression and complex formation of the murine Fanconi anaemia gene product Fancg.

    Science.gov (United States)

    van de Vrugt, Henri J; Koomen, Mireille; Berns, Mariska A D; de Vries, Yne; Rooimans, Martin A; van der Weel, Laura; Blom, Eric; de Groot, Jan; Schepers, Rik J; Stone, Stacie; Hoatlin, Maureen E; Cheng, Ngan Ching; Joenje, Hans; Arwert, Fré

    2002-03-01

    Fanconi anaemia (FA) is an autosomal recessive chromosomal instability disorder. Six distinct FA disease genes have been identified, the products of which function in an integrated pathway that is thought to support a nuclear caretaker function. Comparison of FA gene characteristics in different species may help to unravel the molecular function of the FA pathway. We have cloned the murine homologue of the Fanconi anaemia complementation group G gene, FANCG/XRCC9. The murine Fancg protein shows an 83% similarity to the human protein sequence, and has a predicted molecular weight of 68.5 kDa. Expression of mouse Fancg in human FA-G lymphoblasts fully corrects their cross-linker hypersensitivity. At mRNA and protein levels we detected the co-expression of Fancg and Fanca in murine tissues. In addition, mouse Fancg and Fanca proteins co-purify by immunoprecipitation. Upon transfection into Fanca-deficient mouse embryonic fibroblasts EGFP-Fancg chimeric protein was detectable in the nucleus. We identified a murine cDNA, Fancg, which cross-complements the cellular defect of human FA-G cells and thus represents a true homologue of human FANCG. Spleen, thymus and testis showed the highest Fancg expression levels. Although Fancg and Fanca are able to form a complex, this interaction is not required for Fancg to accumulate in the nuclear compartment.

  20. Heterogeneity of astrocytes: from development to injury - single cell gene expression.

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    Vendula Rusnakova

    Full Text Available Astrocytes perform control and regulatory functions in the central nervous system; heterogeneity among them is still a matter of debate due to limited knowledge of their gene expression profiles and functional diversity. To unravel astrocyte heterogeneity during postnatal development and after focal cerebral ischemia, we employed single-cell gene expression profiling in acutely isolated cortical GFAP/EGFP-positive cells. Using a microfluidic qPCR platform, we profiled 47 genes encoding glial markers and ion channels/transporters/receptors participating in maintaining K(+ and glutamate homeostasis per cell. Self-organizing maps and principal component analyses revealed three subpopulations within 10-50 days of postnatal development (P10-P50. The first subpopulation, mainly immature glia from P10, was characterized by high transcriptional activity of all studied genes, including polydendrocytic markers. The second subpopulation (mostly from P20 was characterized by low gene transcript levels, while the third subpopulation encompassed mature astrocytes (mainly from P30, P50. Within 14 days after ischemia (D3, D7, D14, additional astrocytic subpopulations were identified: resting glia (mostly from P50 and D3, transcriptionally active early reactive glia (mainly from D7 and permanent reactive glia (solely from D14. Following focal cerebral ischemia, reactive astrocytes underwent pronounced changes in the expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and reactive astrocyte markers.

  1. Plant-Derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Naseri, Gita; Balazadeh, Salma; Machens, Fabian; Kamranfar, Iman; Messerschmidt, Katrin; Mueller-Roeber, Bernd

    2017-09-15

    Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.

  2. Polarized two-photon photoselection in EGFP: Theory and experiment.

    Science.gov (United States)

    Masters, T A; Marsh, R J; Blacker, T S; Armoogum, D A; Larijani, B; Bain, A J

    2018-04-07

    In this work, we present a complete theoretical description of the excited state order created by two-photon photoselection from an isotropic ground state; this encompasses both the conventionally measured quadrupolar (K = 2) and the "hidden" degree of hexadecapolar (K = 4) transition dipole alignment, their dependence on the two-photon transition tensor and emission transition dipole moment orientation. Linearly and circularly polarized two-photon absorption (TPA) and time-resolved single- and two-photon fluorescence anisotropy measurements are used to determine the structure of the transition tensor in the deprotonated form of enhanced green fluorescent protein. For excitation wavelengths between 800 nm and 900 nm, TPA is best described by a single element, almost completely diagonal, two-dimensional (planar) transition tensor whose principal axis is collinear to that of the single-photon S 0 → S 1 transition moment. These observations are in accordance with assignments of the near-infrared two-photon absorption band in fluorescent proteins to a vibronically enhanced S 0 → S 1 transition.

  3. Polarized two-photon photoselection in EGFP: Theory and experiment

    Science.gov (United States)

    Masters, T. A.; Marsh, R. J.; Blacker, T. S.; Armoogum, D. A.; Larijani, B.; Bain, A. J.

    2018-04-01

    In this work, we present a complete theoretical description of the excited state order created by two-photon photoselection from an isotropic ground state; this encompasses both the conventionally measured quadrupolar (K = 2) and the "hidden" degree of hexadecapolar (K = 4) transition dipole alignment, their dependence on the two-photon transition tensor and emission transition dipole moment orientation. Linearly and circularly polarized two-photon absorption (TPA) and time-resolved single- and two-photon fluorescence anisotropy measurements are used to determine the structure of the transition tensor in the deprotonated form of enhanced green fluorescent protein. For excitation wavelengths between 800 nm and 900 nm, TPA is best described by a single element, almost completely diagonal, two-dimensional (planar) transition tensor whose principal axis is collinear to that of the single-photon S0 → S1 transition moment. These observations are in accordance with assignments of the near-infrared two-photon absorption band in fluorescent proteins to a vibronically enhanced S0 → S1 transition.

  4. Melittin restores PTEN expression by down-regulating HDAC2 in human hepatocelluar carcinoma HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Hui Zhang

    Full Text Available Melittin is a water-soluble toxic peptide derived from the venom of the bee. Although many studies show the anti-tumor activity of melittin in human cancer including glioma cells, the underlying mechanisms remain elusive. Here the effect of melittin on human hepatocelluar carcinoma HepG2 cell proliferation in vitro and further mechanisms was investigated. We found melittin could inhibit cell proliferation in vitro using Flow cytometry and MTT method. Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4. Results of western Blot and Real-time PCR analysis indicated that melittin was capable to upregulate the expression of PTEN and attenuate histone deacetylase 2 (HDAC2 expression. Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression. Conversely, it was that the potential utility of melittin on PTEN expression was reversed in cells treated with a recombinant pEGFP-C2-HDAC2 plasmid. In addition, treatment with melittin caused a downregulation of Akt phosphorylation, while overexpression of HDAC2 promoted Akt phosphorylation. These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways.

  5. Csf1r-mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System.

    Science.gov (United States)

    Hawley, Catherine A; Rojo, Rocio; Raper, Anna; Sauter, Kristin A; Lisowski, Zofia M; Grabert, Kathleen; Bain, Calum C; Davis, Gemma M; Louwe, Pieter A; Ostrowski, Michael C; Hume, David A; Pridans, Clare; Jenkins, Stephen J

    2018-03-15

    CSF1 is the primary growth factor controlling macrophage numbers, but whether expression of the CSF1 receptor differs between discrete populations of mononuclear phagocytes remains unclear. We have generated a Csf1r -mApple transgenic fluorescent reporter mouse that, in combination with lineage tracing, Alexa Fluor 647-labeled CSF1-Fc and CSF1, and a modified Δ Csf1- enhanced cyan fluorescent protein (ECFP) transgene that lacks a 150 bp segment of the distal promoter, we have used to dissect the differentiation and CSF1 responsiveness of mononuclear phagocyte populations in situ. Consistent with previous Csf1r- driven reporter lines, Csf1r -mApple was expressed in blood monocytes and at higher levels in tissue macrophages, and was readily detectable in whole mounts or with multiphoton microscopy. In the liver and peritoneal cavity, uptake of labeled CSF1 largely reflected transgene expression, with greater receptor activity in mature macrophages than monocytes and tissue-specific expression in conventional dendritic cells. However, CSF1 uptake also differed between subsets of monocytes and discrete populations of tissue macrophages, which in macrophages correlated with their level of dependence on CSF1 receptor signaling for survival rather than degree of transgene expression. A double Δ Csf1r -ECFP- Csf1r -mApple transgenic mouse distinguished subpopulations of microglia in the brain, and permitted imaging of interstitial macrophages distinct from alveolar macrophages, and pulmonary monocytes and conventional dendritic cells. The Csf1r- mApple mice and fluorescently labeled CSF1 will be valuable resources for the study of macrophage and CSF1 biology, which are compatible with existing EGFP-based reporter lines. Copyright © 2018 The Authors.

  6. Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker.

    Science.gov (United States)

    David, Robert; Groebner, Michael; Franz, Wolfgang-Michael

    2005-04-01

    Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (DeltaCD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused DeltaCD4 to an intracellular enhanced green fluorescent protein domain (DeltaCD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of DeltaCD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of DeltaCD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of DeltaCD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.

  7. Adenoviral vectors for highly selective gene expression in central serotonergic neurons reveal quantal characteristics of serotonin release in the rat brain

    Directory of Open Access Journals (Sweden)

    Teschemacher Anja G

    2009-03-01

    Full Text Available Abstract Background 5-hydroxytryptamine (5 HT, serotonin is one of the key neuromodulators in mammalian brain, but many fundamental properties of serotonergic neurones and 5 HT release remain unknown. The objective of this study was to generate an adenoviral vector system for selective targeting of serotonergic neurones and apply it to study quantal characteristics of 5 HT release in the rat brain. Results We have generated adenoviral vectors which incorporate a 3.6 kb fragment of the rat tryptophan hydroxylase-2 (TPH-2 gene which selectively (97% co-localisation with TPH-2 target raphe serotonergic neurones. In order to enhance the level of expression a two-step transcriptional amplification strategy was employed. This allowed direct visualization of serotonergic neurones by EGFP fluorescence. Using these vectors we have performed initial characterization of EGFP-expressing serotonergic neurones in rat organotypic brain slice cultures. Fluorescent serotonergic neurones were identified and studied using patch clamp and confocal Ca2+ imaging and had features consistent with those previously reported using post-hoc identification approaches. Fine processes of serotonergic neurones could also be visualized in un-fixed tissue and morphometric analysis suggested two putative types of axonal varicosities. We used micro-amperometry to analyse the quantal characteristics of 5 HT release and found that central 5 HT exocytosis occurs predominantly in quanta of ~28000 molecules from varicosities and ~34000 molecules from cell bodies. In addition, in somata, we observed a minority of large release events discharging on average ~800000 molecules. Conclusion For the first time quantal release of 5 HT from somato-dendritic compartments and axonal varicosities in mammalian brain has been demonstrated directly and characterised. Release from somato-dendritic and axonal compartments might have different physiological functions. Novel vectors generated in this

  8. Characterization and chondrocyte differentiation stage-specific expression of KRAB zinc-finger protein gene ZNF470

    International Nuclear Information System (INIS)

    Hering, Thomas M.; Kazmi, Najam H.; Huynh, Tru D.; Kollar, John; Xu, Laura; Hunyady, Aaron B.; Johnstone, Brian

    2004-01-01

    As part of a study to identify novel transcriptional regulators of chondrogenesis-related gene expression, we have cloned and characterized cDNA for zinc-finger protein 470 (ZNF470), the human ortholog of which encodes a 717 amino acid residue protein containing 17 Cys 2 His 2 zinc-finger domains, as well as KRAB-A and KRAB-B motifs. The cDNA library used to isolate the initial ZNF470 clone was prepared from human bone marrow-derived mesenchymal progenitor cells at an intermediate stage of chondrogenic differentiation. We have determined the intron-exon structure of the human ZNF470 gene, which has been mapped to a zinc-finger cluster in a known imprinted region of human chromosome 19q13.4. ZNF470 is expressed at high levels in human testis and is expressed at low or undetectible levels in other adult tissues. Human ZNF470 expressed in mammalian cells as an EGFP fusion protein localizes predominantly to the nucleus, consistent with a role in transcriptional regulation. ZNF470, analyzed by quantitative real time PCR, was transiently expressed before the maximal expression of COL2A1 during chondrogenic differentiation in vitro. We have also characterized the bovine ortholog of human ZNF470, which encodes a 508 amino acid residue protein having 10 zinc-finger domains. A bovine ZNF470 cDNA clone was used to examine expression of ZNF470 in bovine articular chondrocytes treated with retinoic acid to stimulate dedifferentiation. Bovine ZNF470 expression was undetectable in freshly isolated bovine articular chondrocytes, but was dramatically upregulated in dedifferentiated retinoic acid-treated chondrocytes. These results, in two model systems, suggest a possible role for ZNF470 in the regulation of chondrogenesis-specific gene expression

  9. FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate

    International Nuclear Information System (INIS)

    Eyre, Nicholas S.; Cleland, Leslie G.; Mayrhofer, Graham

    2008-01-01

    Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. We propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells

  10. Molecular characterization and expression of Rab7 from Clonorchis sinensis and its potential role in autophagy.

    Science.gov (United States)

    Jia, Feifei; Li, Ye; Huang, Yan; Chen, Tingjin; Li, Shan; Xu, Yanquan; Wu, Zhongdao; Li, Xuerong; Yu, Xinbing

    2013-07-01

    Accumulating evidences suggest that Rab7 GTPase is important for the normal progression of autophagy. However, the role of Rab7 GTPase in regulation of autophagy in Clonorchis sinensis is not known. In this study, a gene encoding Rab7 was isolated from C. sinensis adult cDNA. Recombinant CsRab7 was expressed and purified from Escherichia coli. CsRab7 transcripts were detected in the cDNA of adult worm, metacercaria, cercaria, and egg of C. sinensis, and were highly expressed in the metacercaria. Immunohistochemical localization results revealed that CsRab7 was specifically deposited on the vitellarium and eggs of adult worm. Furthermore, EGFP signal of CsRab7WT and the active mutant CsRab7Q67L were associated with autophagic vesicles in transiently transfected 293T cells. It is concluded from the present study that CsRab7 GTPase possibly contributes to the development of C. sinensis and that the autophagy pathway could be an important site of action with respect to the developmental role of CsRab7 in C. sinensis.

  11. Four inducible promoters for controlled gene expression in the oleaginous yeast Rhodotorula toruloides

    Directory of Open Access Journals (Sweden)

    Alexander Michael Bedford Johns

    2016-10-01

    Full Text Available Rhodotorula (Rhodosporidium toruloides is an oleaginous yeast with great biotechnological potential, capable of accumulating lipid up to 70 % of its dry biomass, and of carotenoid biosynthesis. However, few molecular genetic tools are available for manipulation of this basidiomycete yeast and its high genomic GC content can make routine cloning difficult. We have developed plasmid vectors for transformation of R. toruloides which include elements for Saccharomyces cerevisiae in-yeast assembly; this method is robust to the assembly of GC-rich DNA and of large plasmids. Using such vectors we screened for controllable promoters, and identified inducible promoters from the genes NAR1, ICL1, CTR3 and MET16. These four promoters have independent induction/repression conditions and exhibit different levels and rates of induction in R. toruloides, making them appropriate for controllable transgene expression in different experimental situations. Nested deletions were used to identify regulatory regions in the four promoters, and to delimit the minimal inducible promoters, which are as small as 200 bp for the NAR1 promoter. The NAR1 promoter shows very tight regulation under repressed conditions as determined both by an EGFP reporter gene and by conditional rescue of a leu2 mutant. These new tools facilitate molecular genetic manipulation and controllable gene expression in R. toruloides.

  12. Expression and localization of a novel phosducin-like protein from amphioxus Branchiostoma belcheri

    Science.gov (United States)

    Saren, Gaowa; Zhao, Yonggang

    2009-05-01

    A full length amphioxus cDNA, encoding a novel phosducin-like protein ( Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/ Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri.

  13. The establishment of Saccharomyces boulardii surface display system using a single expression vector.

    Science.gov (United States)

    Wang, Tiantian; Sun, Hui; Zhang, Jie; Liu, Qing; Wang, Longjiang; Chen, Peipei; Wang, Fangkun; Li, Hongmei; Xiao, Yihong; Zhao, Xiaomin

    2014-03-01

    In the present study, an a-agglutinin-based Saccharomyces boulardii surface display system was successfully established using a single expression vector. Based on the two protein co-expression vector pSP-G1 built by Partow et al., a S. boulardii surface display vector-pSDSb containing all the display elements was constructed. The display results of heterologous proteins were confirmed by successfully displaying enhanced green fluorescent protein (EGFP) and chicken Eimeria tenella Microneme-2 proteins (EtMic2) on the S. boulardii cell surface. The DNA sequence of AGA1 gene from S. boulardii (SbAGA1) was determined and used as the cell wall anchor partner. This is the first time heterologous proteins have been displayed on the cell surface of S. boulardii. Because S. boulardii is probiotic and eukaryotic, its surface display system would be very valuable, particularly in the development of a live vaccine against various pathogenic organisms especially eukaryotic pathogens such as protistan parasites. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Kisspeptin Activates Ankrd 26 Gene Expression in Migrating Embryonic GnRH Neurons

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    Tomoko eSoga

    2016-03-01

    Full Text Available Kisspeptin, a newly discovered neuropeptide regulates gonadotropin-releasing hormone (GnRH. Kisspeptins are a large RF-amide family of peptides. The kisspeptin coded by kiss1 gene is a 145-amino acid- protein that is cleaved to C-terminal peptide kisspeptin-10. G-protein coupled receptor 54 (GPR54 has been identified as a kisspeptin receptor, and it is expressed in GnRH neurons and in a variety of cancer cells. In this study, enhanced green fluorescent protein (EGFP labelled GnRH cells with migratory properties, which express GPR54, served as a model to study the effects of kisspeptin on cell migration. We monitored EGFP–GnRH neuronal migration in brain slide culture of embryonic day 14 transgenic rat by live cell imaging system and studied the effects of kisspeptin-10 (1nM treatment for 36h on GnRH migration. Furthermore to determine kisspeptin-induced molecular pathways related with apoptosis, and cytoskeletal changes during neuronal migration, we studied the expression levels of candidate genes in laser captured EGFP–GnRH neurons by real time PCR. We found that there was no change in the expression level of genes related to cell proliferation and apoptosis. The expression of ankyrin repeat domain-containing protein (ankrd 26 in EGFP–GnRH neurons was up-regulated by the exposure to kisspeptin. These studies suggest that ankrd26 gene plays an unidentified role in regulating neuronal movement mediated by kisspeptin-GPR54 signaling, which could be a potential pathway to suppress cell migration.

  15. Cell adhesion molecules expression pattern indicates that somatic cells arbitrate gonadal sex of differentiating bipotential fetal mouse gonad.

    Science.gov (United States)

    Piprek, Rafal P; Kolasa, Michal; Podkowa, Dagmara; Kloc, Malgorzata; Kubiak, Jacek Z

    2017-10-01

    Unlike other organ anlagens, the primordial gonad is sexually bipotential in all animals. In mouse, the bipotential gonad differentiates into testis or ovary depending on the genetic sex (XY or XX) of the fetus. During gonad development cells segregate, depending on genetic sex, into distinct compartments: testis cords and interstitium form in XY gonad, and germ cell cysts and stroma in XX gonad. However, our knowledge of mechanisms governing gonadal sex differentiation remains very vague. Because it is known that adhesion molecules (CAMs) play a key role in organogenesis, we suspected that diversified expression of CAMs should also play a crucial role in gonad development. Using microarray analysis we identified 129 CAMs and factors regulating cell adhesion during sexual differentiation of mouse gonad. To identify genes expressed differentially in three cell lines in XY and XX gonads: i) supporting (Sertoli or follicular cells), ii) interstitial or stromal cells, and iii) germ cells, we used transgenic mice expressing EGFP reporter gene and FACS cell sorting. Although a large number of CAMs expressed ubiquitously, expression of certain genes was cell line- and genetic sex-specific. The sets of CAMs differentially expressed in supporting versus interstitial/stromal cells may be responsible for segregation of these two cell lines during gonadal development. There was also a significant difference in CAMs expression pattern between XY supporting (Sertoli) and XX supporting (follicular) cells but not between XY and XX germ cells. This indicates that differential CAMs expression pattern in the somatic cells but not in the germ line arbitrates structural organization of gonadal anlagen into testis or ovary. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Quantitative assessment of fibroblast growth factor receptor 1 expression in neurons and glia

    Directory of Open Access Journals (Sweden)

    Lisha Choubey

    2017-04-01

    Full Text Available Background Fibroblast growth factors (FGFs and their receptors (FGFRs have numerous functions in the developing and adult central nervous system (CNS. For example, the FGFR1 receptor is important for proliferation and fate specification of radial glial cells in the cortex and hippocampus, oligodendrocyte proliferation and regeneration, midline glia morphology and soma translocation, Bergmann glia morphology, and cerebellar morphogenesis. In addition, FGFR1 signaling in astrocytes is required for postnatal maturation of interneurons expressing parvalbumin (PV. FGFR1 is implicated in synapse formation in the hippocampus, and alterations in the expression of Fgfr1 and its ligand, Fgf2 accompany major depression. Understanding which cell types express Fgfr1 during development may elucidate its roles in normal development of the brain as well as illuminate possible causes of certain neuropsychiatric disorders. Methods Here, we used a BAC transgenic reporter line to trace Fgfr1 expression in the developing postnatal murine CNS. The specific transgenic line employed was created by the GENSAT project, tgFGFR1-EGFPGP338Gsat, and includes a gene encoding enhanced green fluorescent protein (EGFP under the regulation of the Fgfr1 promoter, to trace Fgfr1 expression in the developing CNS. Unbiased stereological counts were performed for several cell types in the cortex and hippocampus. Results This model reveals that Fgfr1 is primarily expressed in glial cells, in both astrocytes and oligodendrocytes, along with some neurons. Dual labeling experiments indicate that the proportion of GFP+ (Fgfr1+ cells that are also GFAP+ increases from postnatal day 7 (P7 to 1 month, illuminating dynamic changes in Fgfr1 expression during postnatal development of the cortex. In postnatal neurogenic areas, GFP expression was also observed in SOX2, doublecortin (DCX, and brain lipid-binding protein (BLBP expressing cells. Fgfr1 is also highly expressed in DCX positive cells of

  17. Expression of Two N1 Clones with Single Amino Acid Dissimilarity of Avian Influenza H5N1 Virus

    Directory of Open Access Journals (Sweden)

    RISZA HARTAWAN

    2012-12-01

    Full Text Available Two clones of N1 gene derived from isolate A/Dk/Tangerang/Bbalitvet-ACIAR-TE11/2007 (H5N1 exhibit single mismatch of amino acid sequence at position 242 that is threonine and methionine for the clone #3 and #5, respectively. In order to evaluate the effect of the amino acid substitution, these clones were inserted into two different expression vectors that are pEGFP-C1 and pcDNA-3.3 TOPO® TA cloning. Subsequently, the respective recombinant clones were transfected into eukaryotic cells, including CEF, RK13 and VERO using Lipofectamine ‘plus’ reagent. As a result, the clone #3 retaining atypical sequence showed lower expression level rather than the clone #15 in both vectors and all type of cells. The 3D conformational modelling revealed that the mutation occurs in the inner part of glycoprotein embedded within envelope or matrix. Therefore, the missense mutation seems has no effect on the antigenic properties of neuraminidase but this substitution by any means causes lethal mutagenesis in the individual gene expression by reducing level of protein transcript.

  18. In vitro gene imaging by luciferase to detect the expression and effect of human tumor necrosis factor related apoptosis-inducing ligand in lung cancer A549 cells

    International Nuclear Information System (INIS)

    Zhao Na; Cui Jianling; Guo Zhiyuan; Guo Zhiping; Sun Yingcai; Liu Jicun

    2009-01-01

    Objective: To detect the expression and effect of human tumor necrosis factor related apoptosis-inducing ligand(hTRAIL) in vitro by using a novel double expressing adenoviral vector encoding hTRAIL and firefly luciferase (luc) gene (Ad-hTRAIL-luc), in which luc was used as reporter gene. Methods: A549 cells were transduced with the adenoviral vector encoding enhanced green fluorescent protein (EGFP) gene (Ad-EGFP) at variable multiplicity of infection (MOI). Adenoviral transduction efficiency was determined 48 h later. A549 cells were transduced with Ad-hTRAIL-luc at variable MOI, and the following tests were performed 48h later, respectively: the expressive ratio of hTRAIL and the apoptotic ratio of A549 cells were measured by flow cytometer; counts per minute (cpm) of luminescence were measured by scintillation counters. A549 cells were transduced with Ad-luc at variable MOI, and cpm of luminescence was measured by scintillation counters 48 h later. After A549 cells were transduced with Ad-hTRAIL-luc, the expressive ratio of hTRAIL, the apoptotic ratio of A549 cells and cpm of luminescence were analyzed by one-way ANOVA. The positive ratio of EGFP and cpm of luminescence (Ad-luc) were analyzed by nonparametric ANOVA. Results: After A549 cells were transfected with Ad-hTRAIL-luc, the expressive ratio of hTRAIL on the cell membrane of the groups were (2.37±0.04)%, (3.16±0.03)%, (3.64± 0.03)%, (3.96±0.02)%, (4.24±0.02)%, (4.34±0.02)% respectively, which showed significant difference between each other (P<0.01); and the apoptotic ratio of A549 cells were (1.52±0.04)%, (2.93±0.02)%, (3.39±0.02)%, (3.64±0.02)%, (3.86±0.02)%, (4.08±0.02)%, (4.20± 0.02)%, respectively, and it showed significant difference between each other (P<0.01); cpm of luminescence were 465 561 ± 26 801, 1 038 576 ± 29 417, 937 655 ± 23 197, 786 432 ± 20 028, 524 288 ± 16 338, 401 566 ± 15 961, respectively, and it also showed significant difference between each other (P<0

  19. Molecular fingerprint of neuropeptide S-producing neurons in the mouse brain

    DEFF Research Database (Denmark)

    Liu, Xiaobin; Zeng, Joanne; Zhou, Anni

    2011-01-01

    /EGFP-transgenic mice show anatomically correct and overlapping expression of both NPS and EGFP. A total number of ~500 NPS/EGFP-positive neurons are present in the mouse brain, located in the pericoerulear region and the Kölliker-Fuse nucleus. NPS and transgene expression is first detectable around E14, indicating...

  20. Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris.

    Science.gov (United States)

    Vogl, Thomas; Gebbie, Leigh; Palfreyman, Robin W; Speight, Robert

    2018-03-15

    Pichia pastoris (syn. Komagataella phaffii ) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae , where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs). IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast

  1. Conditional and inducible transgene expression in endothelial and hematopoietic cells using Cre/loxP and tetracycline-off systems.

    Science.gov (United States)

    Liu, Ju; Deutsch, Urban; Fung, Iris; Lobe, Corrinne G

    2014-11-01

    In the present study, the tetracycline-off and Cre/ loxP systems were combined to gain temporal and spatial control of transgene expression. Mice were generated that carried three transgenes: Tie2-tTA, tet-O-Cre and either the ZEG or ZAP reporter. Tie2-tTA directs expression of tetracycline-controlled transactivator (tTA) in endothelial and hematopoietic cells under the control of the Tie2 promoter. Tet-O-Cre produces Cre recombinase from a minimal promoter containing the tet-operator (tetO). ZEG or ZAP contains a strong promoter and a loxP -flanked stop sequence, followed by an enhanced green fluorescence protein (EGFP) or human placental alkaline phosphatase (hPLAP) reporter. In the presence of tetracycline, the tTA transactivator produced by Tie-2-tTA is disabled and Cre is not expressed. In the absence of tetracycline, the tTA binds tet-O-Cre to drive the expression of Cre, which recombines the loxP sites of the ZEG or ZAP transgene and results in reporter gene expression. In the present study, the expression of the ZEG or ZAP reporter genes in embryos and adult animals with and without tetracycline treatment was examined. In the presence of tetracycline, no reporter gene expression was observed. When tetracycline was withdrawn, Cre excision was activated and the reporter genes were detected in endothelial and hematopoietic cells. These results demonstrate that this system may be used to bypass embryonic lethality and access adult phenotypes.

  2. Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function.

    Directory of Open Access Journals (Sweden)

    Cheng-Tao Yang

    2009-07-01

    Full Text Available The pH and voltage-regulated Slo3 K(+ channel, a homologue of the Ca(2+- and voltage-regulated Slo1 K(+ channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary beta subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels.To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 subunit produced an 8-10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm.These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 alpha subunit.

  3. Intrathecal long-term gene expression by self-complementary adeno-associated virus type 1 suitable for chronic pain studies in rats

    Directory of Open Access Journals (Sweden)

    Janssen William GM

    2006-01-01

    Full Text Available Abstract Background Intrathecal (IT gene transfer is an attractive approach for targeting spinal mechanisms of nociception but the duration of gene expression achieved by reported methods is short (up to two weeks impairing their utility in the chronic pain setting. The overall goal of this study was to develop IT gene transfer yielding true long-term transgene expression defined as ≥ 3 mo following a single vector administration. We defined "IT" administration as atraumatic injection into the lumbar cerebrospinal fluid (CSF modeling a lumbar puncture. Our studies focused on recombinant adeno-associated virus (rAAV, one of the most promising vector types for clinical use. Results Conventional single stranded rAAV2 vectors performed poorly after IT delivery in rats. Pseudotyping of rAAV with capsids of serotypes 1, 3, and 5 was tested alone or in combination with a modification of the inverted terminal repeat. The former alters vector tropism and the latter allows packaging of self-complementary rAAV (sc-rAAV vectors. Combining both types of modification led to the identification of sc-rAAV2/l as a vector that performed superiorly in the IT space. IT delivery of 3 × 10e9 sc-rAAV2/l particles per animal led to stable expression of enhanced green fluorescent protein (EGFP for ≥ 3 mo detectable by Western blotting, quantitative PCR, and in a blinded study by confocal microscopy. Expression was strongest in the cauda equina and the lower sections of the spinal cord and only minimal in the forebrain. Microscopic examination of the SC fixed in situ with intact nerve roots and meninges revealed strong EGFP fluorescence in the nerve roots. Conclusion sc-rAAVl mediates stable IT transgene expression for ≥ 3 mo. Our findings support the underlying hypothesis that IT target cells for gene transfer lack the machinery for efficient conversion of the single-stranded rAAV genome into double-stranded DNA and favor uptake of serotype 1 vectors over 2

  4. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

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    Haohan Wang

    2016-03-01

    Full Text Available Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway.

  5. Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos

    Directory of Open Access Journals (Sweden)

    Lee Okhyun

    2012-06-01

    Full Text Available Abstract Background Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38 in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff, contains three copies of oestrogen response elements (3ERE that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein. Results The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2, the synthetic oestrogen 17α- ethinyloestradiol (EE2, and the relatively weak environmental oestrogen nonylphenol (NP, and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures. For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish. We

  6. Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells

    Science.gov (United States)

    Miura, Hirohito; Scott, Jennifer K.; Harada, Shuitsu; Barlow, Linda A.

    2014-01-01

    Background Taste buds contain ~60 elongate cells and several basal cells. Elongate cells comprise three functional taste cell types: I - glial cells, II - bitter/sweet/umami receptor cells, and III - sour detectors. Although taste cells are continuously renewed, lineage relationships among cell types are ill-defined. Basal cells have been proposed as taste bud stem cells, a subset of which express Sonic hedgehog (Shh). However, Shh+ basal cells turnover rapidly suggesting that Shh+ cells are precursors of some or all taste cell types. Results To fate map Shh-expressing cells, mice carrying ShhCreERT2 and a high (CAG-CAT-EGFP) or low (R26RLacZ) efficiency reporter allele were given tamoxifen to activate Cre in Shh+ cells. Using R26RLacZ, lineage-labeled cells occur singly within buds, supporting a post-mitotic state for Shh+ cells. Using either reporter, we show that Shh+ cells differentiate into all three taste cell types, in proportions reflecting cell type ratios in taste buds (I > II > III). Conclusions Shh+ cells are not stem cells, but are post-mitotic, immediate precursors of taste cells. Shh+ cells differentiate into each of the three taste cell types, and the choice of a specific taste cell fate is regulated to maintain the proper ratio within buds. PMID:24590958

  7. Express web application development

    CERN Document Server

    Yaapa, Hage

    2013-01-01

    Express Web Application Development is a practical introduction to learning about Express. Each chapter introduces you to a different area of Express, using screenshots and examples to get you up and running as quickly as possible.If you are looking to use Express to build your next web application, ""Express Web Application Development"" will help you get started and take you right through to Express' advanced features. You will need to have an intermediate knowledge of JavaScript to get the most out of this book.

  8. The targeting expression of the vascular endothelial growth factor gene in endothelial cells regulated by HRE.ppET-1.

    Science.gov (United States)

    Zheng, Xiangrong; Zhang, Shangshang; Yang, Yujia; Wang, Xia; Zhong, Le; Yu, Xiaohe

    2008-11-01

    The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 mumol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The expression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regulated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (PHRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation of endothelial cells. Taking advantage of these facts could greatly improve the efficiency of gene therapy. The vector would be valuable for various gene transfer

  9. After Effects expressions

    CERN Document Server

    Geduld, Marcus

    2013-01-01

    Put the power of Expressions to work in your animations with controls and efficiencies impossible to achieve with traditional keyframing techniques. No programming skills are required. Foundation concepts and skills orient the new designer and serve as a handy reference to the experienced one. Basics of creating expressions, variables, commands, and expression helpers precede the leap into javascript and math essentials for more advanced expressions that include randomness, physical simularions and 3D. Full color illustrations display the scripts and the resulti

  10. Tumor cell adhesion to endothelial cells is increased by endotoxin via an upregulation of beta-1 integrin expression.

    LENUS (Irish Health Repository)

    Andrews, E J

    2012-02-03

    BACKGROUND: Recent studies have demonstrated that metastatic disease develops from tumor cells that adhere to endothelial cells and proliferate intravascularly. The beta-1 integrin family and its ligand laminin have been shown to be important in tumor-to-endothelial cell adhesion. Lipopolysaccharide (LPS) has been implicated in the increased metastatic tumor growth that is seen postoperatively. We postulated that LPS increases tumor cell expression of beta-1 integrins and that this leads to increased adhesion. METHODS: The human metastatic colon cancer cell line LS174T was labeled with an enhanced green fluorescent protein (eGFP) using retroviral transfection. Cell cultures were treated with LPS for 1, 2, and 4 h (n = 6 each) and were subsequently cocultured for 30 or 120 min with confluent human umbilical vein endothelial cells (HUVECs), to allow adherence. Adherent tumor cells were counted using fluorescence microscopy. These experiments were carried out in the presence or absence of a functional blocking beta-1 integrin monoclonal antibody (4B4). Expression of beta-1 integrin and laminin on tumor and HUVECs was assessed using flow cytometric analysis. Tumor cell NF-kappaB activation after incubation with LPS was measured. RESULTS: Tumor cell and HUVEC beta-1 integrin expression and HUVEC expression of laminin were significantly (P < 0.05) enhanced after incubation with LPS. Tumor cell adhesion to HUVECs was significantly increased. Addition of the beta-1 integrin blocking antibody reduced tumor cell adhesion to control levels. LPS increased tumor cell NF-kappaB activation. CONCLUSIONS: Exposure to LPS increases tumor cell adhesion to the endothelium through a beta-1 integrin-mediated pathway that is NF-kappaB dependent. This may provide a target for immunotherapy directed at reducing postoperative metastatic tumor growth.

  11. Neuroglobin over expressing mice

    DEFF Research Database (Denmark)

    Raida, Zindy; Hundahl, Christian Ansgar; Nyengaard, Jens R

    2013-01-01

    showed over expression to be confined to primarily the cortex, hippocampus, cerebellum, and only in neurons. The level and expression pattern of endogenous Neuroglobin was unaffected by insertion of the over expressing Ngb transgene. Neuroglobin over expression resulted in a significant reduction...... previous reports, Neuroglobin over expression is not global but confined to a few well-defined brain regions, and only in neurons. This study confirms previous reports showing a correlation between reduced infarct volume and elevated Neuroglobin levels, but underlines the need to study the likely...

  12. Targeted inhibition of osteosarcoma tumor growth by bone marrow-derived mesenchymal stem cells expressing cytosine deaminase/5-fluorocytosine in tumor-bearing mice.

    Science.gov (United States)

    NguyenThai, Quynh-Anh; Sharma, Neelesh; Luong, Do Huynh; Sodhi, Simrinder Singh; Kim, Jeong-Hyun; Kim, Nameun; Oh, Sung-Jong; Jeong, Dong Kee

    2015-01-01

    Mesenchymal stem cells (MSCs) are considered as an attractive approach for gene or drug delivery in cancer therapy. In the present study, the ability of human bone marrow-derived MSCs expressing the cytosine deaminase/5-fluorocytosine prodrug (CD/5-FC MSCs) to target the human osteosarcoma cell line Cal72 was evaluated. The stable CD/5-FC MSC cell line was established by transfection of pEGFP containing the cytosine deaminase gene into MSCs with G418 selection. The anti-tumor effect was verified by a bystander effect assay in vitro and co-injection of Cal72 and CD/5-FC MSCs in cancer-bearing mice. The therapeutic CD/5-FC MSCs retained the characteristics of multipotent cells, such as differentiation into adipocytes/osteocytes and expression of mesenchymal markers (CD90 and CD44), and showed migration toward Cal72 cells to a greater extent than the native MSCs. The bystander effect assay showed that the CD/5-FC MSCs significantly augmented Cal72 cytotoxicity in direct co-culture and in the presence of 5-FC through the application of conditioned medium. In osteosarcoma-bearing mice, the CD/5-FC MSCs inhibited tumor growth compared to control mice subcutaneously injected with only Cal72 cells. Taken together, these findings suggest that CD/5-FC MSCs may be suitable for targeting human osteosarcoma. Copyright © 2015 John Wiley & Sons, Ltd.

  13. Expression, purification, crystallization and preliminary X-ray crystallographic studies of hepatitis B virus core fusion protein corresponding to octahedral particles

    International Nuclear Information System (INIS)

    Kikuchi, Masaki; Iwabuchi, Shinichiro; Kikkou, Tatsuhiko; Noguchi, Keiichi; Odaka, Masafumi; Yohda, Masafumi; Kawata, Masaaki; Sato, Chikara; Matsumoto, Osamu

    2013-01-01

    Novel hepatitis B virus-like particles of recombinant dimeric core–GFP fusion protein were expressed, purified and crystallized. The crystals diffracted to 2.15 Å resolution and belonged to space group F432, with unit-cell parameters a = b = c = 219.7 Å. Recombinant hepatitis B virus core proteins dimerize to form building blocks that are capable of self-assembly into a capsid. A core capsid protein dimer (CPD) linked to a green fluorescent protein variant, EGFP, at the C-terminus has been designed. The recombinant fusion CPD was expressed in Escherichia coli, assembled into virus-like particles (VLPs), purified and crystallized. The single crystal diffracted to 2.15 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 219.7 Å. The fusion proteins assembled into icosahedral VLPs in aqueous solution, but were rearranged into octahedral symmetry through the crystal-packing process under the crystallization conditions

  14. Lactococcus lactis expressing either Staphylococcus aureus fibronectin-binding protein A or Listeria monocytogenes internalin A can efficiently internalize and deliver DNA in human epithelial cells.

    Science.gov (United States)

    Innocentin, Silvia; Guimarães, Valeria; Miyoshi, Anderson; Azevedo, Vasco; Langella, Philippe; Chatel, Jean-Marc; Lefèvre, François

    2009-07-01

    Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA(+)), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA(+) and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA(+)) or its fibronectin binding domains C and D (L. lactis CD(+)). L. lactis FnBPA(+) and L. lactis InlA(+) showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD(+) was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA(+) or L. lactis InlA(+). Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.

  15. Regular Expression Pocket Reference

    CERN Document Server

    Stubblebine, Tony

    2007-01-01

    This handy little book offers programmers a complete overview of the syntax and semantics of regular expressions that are at the heart of every text-processing application. Ideal as a quick reference, Regular Expression Pocket Reference covers the regular expression APIs for Perl 5.8, Ruby (including some upcoming 1.9 features), Java, PHP, .NET and C#, Python, vi, JavaScript, and the PCRE regular expression libraries. This concise and easy-to-use reference puts a very powerful tool for manipulating text and data right at your fingertips. Composed of a mixture of symbols and text, regular exp

  16. Regular expressions cookbook

    CERN Document Server

    Goyvaerts, Jan

    2009-01-01

    This cookbook provides more than 100 recipes to help you crunch data and manipulate text with regular expressions. Every programmer can find uses for regular expressions, but their power doesn't come worry-free. Even seasoned users often suffer from poor performance, false positives, false negatives, or perplexing bugs. Regular Expressions Cookbook offers step-by-step instructions for some of the most common tasks involving this tool, with recipes for C#, Java, JavaScript, Perl, PHP, Python, Ruby, and VB.NET. With this book, you will: Understand the basics of regular expressions through a

  17. The oligodendroglial precursor cell line Oli-neu represents a cell culture system to examine functional expression of the mouse gap junction gene connexin29 (Cx29

    Directory of Open Access Journals (Sweden)

    Goran Christoph Söhl

    2013-06-01

    Full Text Available The potential gap junction forming mouse connexin29 (Cx29 protein is concomitantly expressed with connexin32 (Cx32 in peripheral myelin forming Schwann cells and together with both Cx32 and connexin47 (Cx47 in oligodendrocytes of the CNS. To study the genomic structure and functional expression of Cx29, either primary cells or cell culture systems might be selected, from which the latter are easier to cultivate. Both structure and expression of Cx29 is still not fully understood. In the mouse sciatic nerve, brain and the oligodendroglial precursor cell line Oli-neu the Cx29 gene is processed in two transcript isoforms both harbouring a unique reading frame. In contrast to Cx32 and Cx47, only Cx29 protein is abundantly expressed in undifferentiated as well as differentiated Oli-neu cells but the absence of Etbr dye transfer after microinjection concealed the function of Cx29 mediated gap junction communication between those cells. Although HeLa cells stably transfected with Cx29 or Cx29-eGFP neither demonstrated any permeability for Lucifer yellow nor for neurobiotin, blocking of Etbr uptake from the media by gap junction blockers does suppose a role of Cx29 in hemi-channel function. Thus, we conclude that, due to its high abundance of Cx29 expression and its reproducible culture conditions, the oligodendroglial precursor cell line Oli-neu might constitute an appropriate cell culture system to study molecular mechanisms or putative extracellular stimuli to functionally open Cx29 channels or hemi-channels.

  18. Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo

    International Nuclear Information System (INIS)

    Anesti, Anna-Maria; Simpson, Guy R; Price, Toby; Pandha, Hardev S; Coffin, Robert S

    2010-01-01

    Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEX GM-CSF , we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV. To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and β-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting. Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective. This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials

  19. Darwin and Emotion Expression

    Science.gov (United States)

    Hess, Ursula; Thibault, Pascal

    2009-01-01

    In his book "The Expression of the Emotions in Man and Animals," Charles Darwin (1872/1965) defended the argument that emotion expressions are evolved and adaptive (at least at some point in the past) and serve an important communicative function. The ideas he developed in his book had an important impact on the field and spawned rich domains of…

  20. Freedom of Expression.

    Science.gov (United States)

    Update on Law-Related Education, 1988

    1988-01-01

    Presents an activity which uses hypothetical situations to explore the proper boundaries of freedom of expression and the role of the U.S. Supreme Court in interpreting its limits. Appropriate for grades 4-12, the lesson includes such topics as the "clear and present danger" clause, student expression, obscenity, and defamation. (GEA)

  1. Caricaturing facial expressions.

    Science.gov (United States)

    Calder, A J; Rowland, D; Young, A W; Nimmo-Smith, I; Keane, J; Perrett, D I

    2000-08-14

    The physical differences between facial expressions (e.g. fear) and a reference norm (e.g. a neutral expression) were altered to produce photographic-quality caricatures. In Experiment 1, participants rated caricatures of fear, happiness and sadness for their intensity of these three emotions; a second group of participants rated how 'face-like' the caricatures appeared. With increasing levels of exaggeration the caricatures were rated as more emotionally intense, but less 'face-like'. Experiment 2 demonstrated a similar relationship between emotional intensity and level of caricature for six different facial expressions. Experiments 3 and 4 compared intensity ratings of facial expression caricatures prepared relative to a selection of reference norms - a neutral expression, an average expression, or a different facial expression (e.g. anger caricatured relative to fear). Each norm produced a linear relationship between caricature and rated intensity of emotion; this finding is inconsistent with two-dimensional models of the perceptual representation of facial expression. An exemplar-based multidimensional model is proposed as an alternative account.

  2. Materiality for Musical Expressions

    DEFF Research Database (Denmark)

    Lindell, Rikard; Tahiroğlu, Koray; Riis, Morten S.

    2016-01-01

    Nordic universities. Electronic music instrument makers participated in providing the course. In eleven days the students designed and built interfaces for musical expressions , composed a piece, and performed at the Norberg electronic music festival. The students explored the relationship between......We organised an elven day intense course in materiality for musical expressions to explore underlying principles of New Interfaces for Musical Expression (NIME) in higher education. We grounded the course in different aspects of ma-teriality and gathered interdisciplinary student teams from three...... technology and possible musical expression with a strong connection to culture and place. The emphasis on performance provided closure and motivated teams to move forward in their design and artistic processes. On the basis of the course we discuss an interdisciplinary NIME course syllabus, and we infer...

  3. Localizing Expression of Ambiguity

    National Research Council Canada - National Science Library

    Bear, John; Hobbs, Sr, Jerry R

    1987-01-01

    In this paper we describe an implemented program for localizing the expression of many types of syntactic ambiguity, in the logical forms of sentences, in a manner convenient for subsequent inferential processing...

  4. Gene Expression Omnibus (GEO)

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene Expression Omnibus is a public functional genomics data repository supporting MIAME-compliant submissions of array- and sequence-based data. Tools are provided...

  5. Express.js blueprints

    CERN Document Server

    Augarten, Ben; Lin, Eric; Shaikh, Aidha; Soriani, Fabiano Pereira; Tisserand, Geoffrey; Zhang, Chiqing; Zhang, Kan

    2015-01-01

    This book is for beginners to Node.js and also for those who are technically advanced. By the end of this book, every competent developer will have achieved expertise in building web applications with Express.js.

  6. [Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System].

    Science.gov (United States)

    Li, Lili; Wang, Zhan; Zhou, Yubai; Zhang, Fang; Shen, Sisi; Li, Zelin; Zeng, Yi

    2015-09-01

    For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.

  7. Lamprey Prohibitin2 Arrest G2/M Phase Transition of HeLa Cells through Down-regulating Expression and Phosphorylation Level of Cell Cycle Proteins.

    Science.gov (United States)

    Shi, Ying; Guo, Sicheng; Wang, Ying; Liu, Xin; Li, Qingwei; Li, Tiesong

    2018-03-02

    Prohibitin 2(PHB2) is a member of the SFPH trans-membrane family proteins. It is a highly conserved and functionally diverse protein that plays an important role in preserving the structure and function of the mitochondria. In this study, the lamprey PHB2 gene was expressed in HeLa cells to investigate its effect on cell proliferation. The effect of Lm-PHB2 on the proliferation of HeLa cells was determined by treating the cells with pure Lm-PHB2 protein followed by MTT assay. Using the synchronization method with APC-BrdU and PI double staining revealed rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase. Similarly, cells transfected with pEGFP-N1-Lm-PHB2 also exhibited remarkable reduction in proliferation. Western blot and quantitative real-time PCR(qRT-PCR) assays suggested that Lm-PHB2 caused cell cycle arrest in HeLa cells through inhibition of CDC25C and CCNB1 expression. According to our western blot analysis, Lm-PHB2 was also found to reduce the expression level of Wee1 and PLK1 and the phosphorylation level of CCNB1, CDC25C and CDK1 in HeLa cells. Lamprey prohibitin 2 could arrest G2/M phase transition of HeLa cells through down-regulating expression and phosphorylation level of cell cycle proteins.

  8. Defining Optimized Properties of Modified mRNA to Enhance Virus- and DNA- Independent Protein Expression in Adult Stem Cells and Fibroblasts

    Directory of Open Access Journals (Sweden)

    Frauke Hausburg

    2015-02-01

    Full Text Available Background: By far, most strategies for cell reprogramming and gene therapy are based on the introduction of DNA after viral delivery. To avoid the high risks accompanying these goals, non-viral and DNA-free delivery methods for various cell types are required. Methods: Relying on an initially established PCR-based protocol for convenient template DNA production, we synthesized five differently modified EGFP mRNA (mmRNA species, incorporating various degrees of 5-methylcytidine-5'-triphosphate (5mC and pseudouridine-5'-triphosphate (Ψ. We then investigated their effect on i protein expression efficiencies and ii cell viability for human mesenchymal stem cells (hMSCs and fibroblasts from different origins. Results: Our protocol allows highly efficient mmRNA production in vitro, enabling rapid and stable protein expression after cell transfection. However, our results also demonstrate that the terminally optimal modification needs to be defined in pilot experiments for each particular cell type. Transferring our approach to the conversion of fibroblasts into skeletal myoblasts using mmRNA encoding MyoD, we confirm the huge potential of mmRNA based protein expression for virus- and DNA-free reprogramming strategies. Conclusion: The achieved high protein expression levels combined with good cell viability not only in fibroblasts but also in hMSCs provides a promising option for mmRNA based modification of various cell types including slowly proliferating adult stem cells. Therefore, we are confident that our findings will substantially contribute to the improvement of efficient cell reprogramming and gene therapy approaches.

  9. The expressions of emotions

    DEFF Research Database (Denmark)

    Vishnivetz, Berta

    Abstract On the broadness of the vast field called “Expressions of Emotions” this study focuses on the whole bodily emotional expression. The main question posed is: Whether there are movement patterns specific to each emotion?. I carried out a thorough review of the theories of emotion...... and of expressions of emotions and movement notation that provided the sources for a careful research plan for the empirical process of this study. On this basis I chose to record onto video the four previously choreographed movements that I considered to correspond each of the following emotions: joy, fear, sadness......, anger. The selection of these four emotions demanded previously to clear up the problems the above named survey ensued. When researchers want to describe a certain movement in the field of psychology and non-verbal communication, it may result in disagreements and misunderstandings which sometimes lead...

  10. Freedom of Expression

    Directory of Open Access Journals (Sweden)

    Guilherme Canela

    2007-06-01

    Full Text Available The freedoms of expression and of the press are basic pillars of the western democracies. The contemporary theoretical framework which gives support to these rights was generated in the wake of the liberal revolutions which took place in Western Europe and in North America starting from the second half of the 1600s. Our purpose in this text is to present the current scene regarding this topic, focusing whenever pertinent on the Brazilian case, and seeking to question the unconditional defense of the freedoms of expression and of the press made by the thinkers who founded these principles vis-á-vis contemporary issues of the communicational universe. Going beyond theoretical-conceptual refl ections, we present and analyze the results of a content analysis showing how 53 Brazilian newspapers and 4 magazines with nationwide circulation report (or not topics relating to freedom of expression and of the press.

  11. Regular expression containment

    DEFF Research Database (Denmark)

    Henglein, Fritz; Nielsen, Lasse

    2011-01-01

    We present a new sound and complete axiomatization of regular expression containment. It consists of the conventional axiomatiza- tion of concatenation, alternation, empty set and (the singleton set containing) the empty string as an idempotent semiring, the fixed- point rule E* = 1 + E × E......* for Kleene-star, and a general coin- duction rule as the only additional rule. Our axiomatization gives rise to a natural computational inter- pretation of regular expressions as simple types that represent parse trees, and of containment proofs as coercions. This gives the axiom- atization a Curry......-Howard-style constructive interpretation: Con- tainment proofs do not only certify a language-theoretic contain- ment, but, under our computational interpretation, constructively transform a membership proof of a string in one regular expres- sion into a membership proof of the same string in another regular expression. We...

  12. In Silico Expression Analysis.

    Science.gov (United States)

    Bolívar, Julio; Hehl, Reinhard; Bülow, Lorenz

    2016-01-01

    Information on the specificity of cis-sequences enables the design of functional synthetic plant promoters that are responsive to specific stresses. Potential cis-sequences may be experimentally tested, however, correlation of genomic sequence with gene expression data enables an in silico expression analysis approach to bioinformatically assess the stress specificity of candidate cis-sequences prior to experimental verification. The present chapter demonstrates an example for the in silico validation of a potential cis-regulatory sequence responsive to cold stress. The described online tool can be applied for the bioinformatic assessment of cis-sequences responsive to most abiotic and biotic stresses of plants. Furthermore, a method is presented based on a reverted in silico expression analysis approach that predicts highly specific potentially functional cis-regulatory elements for a given stress.

  13. Designing Emotionally Expressive Robots

    DEFF Research Database (Denmark)

    Tsiourti, Christiana; Weiss, Astrid; Wac, Katarzyna

    2017-01-01

    Socially assistive agents, be it virtual avatars or robots, need to engage in social interactions with humans and express their internal emotional states, goals, and desires. In this work, we conducted a comparative study to investigate how humans perceive emotional cues expressed by humanoid...... robots through five communication modalities (face, head, body, voice, locomotion) and examined whether the degree of a robot's human-like embodiment affects this perception. In an online survey, we asked people to identify emotions communicated by Pepper -a highly human-like robot and Hobbit – a robot...... for robots....

  14. CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.

    Science.gov (United States)

    Yuen, Garmen; Khan, Fehad J; Gao, Shaojian; Stommel, Jayne M; Batchelor, Eric; Wu, Xiaolin; Luo, Ji

    2017-11-16

    CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  15. Mining frequent binary expressions

    NARCIS (Netherlands)

    Calders, T.; Paredaens, J.; Kambayashi, Y.; Mohania, M.K.; Tjoa, A.M.

    2000-01-01

    In data mining, searching for frequent patterns is a common basic operation. It forms the basis of many interesting decision support processes. In this paper we present a new type of patterns, binary expressions. Based on the properties of a specified binary test, such as reflexivity, transitivity

  16. Facial Expression Recognition

    NARCIS (Netherlands)

    Pantic, Maja; Li, S.; Jain, A.

    2009-01-01

    Facial expression recognition is a process performed by humans or computers, which consists of: 1. Locating faces in the scene (e.g., in an image; this step is also referred to as face detection), 2. Extracting facial features from the detected face region (e.g., detecting the shape of facial

  17. Sensual expressions on faces

    NARCIS (Netherlands)

    Hendriks, A.W.C.J.; Engels, R.C.M.E.; Roek, M.A.E.

    2009-01-01

    We explored the possibility that an emotional facial expression exists specifically for signalling sexual interest. We selected photographs of twenty-eight fashion models (male and female) with large portfolios (range 81 - 1593), choosing only face photographs in which the model was looking into the

  18. Experience and Expression

    Science.gov (United States)

    Hanes, Jay Michael; Weisman, Eleanor

    2016-01-01

    Two artist-educators analyzed their creative process informed by John Dewey's concepts regarding the act of expression. The essay interweaves a description of their performance piece with a discussion of conceptual processes, including intermediality and collaboration as crucial in art making, learning, and pedagogical efficacy. Both the creation…

  19. The structural determinants responsible for c-Fos protein proteasomal degradation differ according to the conditions of expression.

    Science.gov (United States)

    Ferrara, Patrizia; Andermarcher, Elisabetta; Bossis, Guillaume; Acquaviva, Claire; Brockly, Frédérique; Jariel-Encontre, Isabelle; Piechaczyk, Marc

    2003-03-13

    c-fos gene is expressed constitutively in a number of tissues as well as in certain tumor cells and is inducible, in general rapidly and transiently, in virtually all other cell types by a variety of stimuli. Its protein product, c-Fos, is a short-lived transcription factor that heterodimerizes with various protein partners within the AP-1 transcription complex via leucine zipper/leucine zipper interactions for binding to specific DNA sequences. It is mostly, if not exclusively, degraded by the proteasome. To localize the determinant(s) responsible for its instability, we have conducted a genetic analysis in which the half-lives of c-Fos mutants and chimeras made with the stable EGFP reporter protein were compared under two experimental conditions taken as example of continous and inducible expression. Those were constitutive expression in asynchronously growing Balb/C 3T3 mouse embryo fibroblasts and transient induction in the same cells undergoing the G0/G1 phase transition upon stimulation by serum. Our work shows that c-Fos is degraded faster in synchronous- than in asynchronous cells. This difference in turnover is primarily accounted for by several mechanisms. First, in asynchronous cells, a unique C-terminal destabilizer is active whereas, in serum-stimulated cells two destabilizers located at both extremities of the protein are functional. Second, heterodimerization and/or binding to DNA accelerates protein degradation only during the G0/G1 phase transition. Adding another level of complexity to turnover control, phosphorylation at serines 362 and 374, which are c-Fos phosphorylation sites largely modified during the G0/G1 phase transition, stabilizes c-Fos much more efficiently in asynchronous than in serum-stimulated cells. In both cases, the reduced degradation rate is due to inhibition of the activity of the C-terminal destabilizer. However, in serum-stimulated cells, this effect is partially masked by the activation of the N-terminal destabilizer and

  20. Facial expressions recognition with an emotion expressive robotic head

    Science.gov (United States)

    Doroftei, I.; Adascalitei, F.; Lefeber, D.; Vanderborght, B.; Doroftei, I. A.

    2016-08-01

    The purpose of this study is to present the preliminary steps in facial expressions recognition with a new version of an expressive social robotic head. So, in a first phase, our main goal was to reach a minimum level of emotional expressiveness in order to obtain nonverbal communication between the robot and human by building six basic facial expressions. To evaluate the facial expressions, the robot was used in some preliminary user studies, among children and adults.

  1. Transmembrane and ubiquitin-like domain-containing protein 1 (Tmub1/HOPS facilitates surface expression of GluR2-containing AMPA receptors.

    Directory of Open Access Journals (Sweden)

    Hyunjeong Yang

    Full Text Available Some ubiquitin-like (UBL domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported.Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1 protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.

  2. Expression profiling of migrated and invaded breast cancer cells predicts early metastatic relapse and reveals Krüppel-like factor 9 as a potential suppressor of invasive growth in breast cancer

    Science.gov (United States)

    Limame, Ridha; de Beeck, Ken Op; Van Laere, Steven; Croes, Lieselot; De Wilde, Annemieke; Dirix, Luc; Van Camp, Guy; Peeters, Marc; De Wever, Olivier; Lardon, Filip; Pauwels, Patrick

    2014-01-01

    Cell motility and invasion initiate metastasis. However, only a subpopulation of cancer cells within a tumor will ultimately become invasive. Due to this stochastic and transient nature, in an experimental setting, migrating and invading cells need to be isolated from the general population in order to study the gene expression profiles linked to these processes. This report describes microarray analysis on RNA derived from migrated or invaded subpopulations of triple negative breast cancer cells in a Transwell set-up, at two different time points during motility and invasion, pre-determined as “early” and “late” in real-time kinetic assessments. Invasion- and migration-related gene expression signatures were generated through comparison with non-invasive cells, remaining at the upper side of the Transwell membranes. Late-phase signatures of both invasion and migration indicated poor prognosis in a series of breast cancer data sets. Furthermore, evaluation of the genes constituting the prognostic invasion-related gene signature revealed Krüppel-like factor 9 (KLF9) as a putative suppressor of invasive growth in breast cancer. Next to loss in invasive vs non-invasive cell lines, KLF9 also showed significantly lower expression levels in the “early” invasive cell population, in several public expression data sets and in clinical breast cancer samples when compared to normal tissue. Overexpression of EGFP-KLF9 fusion protein significantly altered morphology and blocked invasion and growth of MDA-MB-231 cells in vitro. In addition, KLF9 expression correlated inversely with mitotic activity in clinical samples, indicating anti-proliferative effects. PMID:25593984

  3. Recombinant canine distemper virus strain Snyder Hill expressing green or red fluorescent proteins causes meningoencephalitis in the ferret.

    Science.gov (United States)

    Ludlow, M; Nguyen, D T; Silin, D; Lyubomska, O; de Vries, R D; von Messling, V; McQuaid, S; De Swart, R L; Duprex, W P

    2012-07-01

    The propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDV(SH)) and show that this virus rapidly circumvents the blood-brain and blood-cerebrospinal fluid (CSF) barriers to spread into the subarachnoid space to induce dramatic viral meningoencephalitis. The use of recombinant CDV(SH) (rCDV(SH)) expressing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dTomato) facilitated the sensitive pathological assessment of routes of virus spread in vivo. Infection of ferrets with these viruses led to the full spectrum of clinical signs typically associated with distemper in dogs during a rapid, fatal disease course of approximately 2 weeks. Comparison with the ferret-adapted CDV(5804P) and the prototypic wild-type CDV(R252) showed that hematogenous infection of the choroid plexus is not a significant route of virus spread into the CSF. Instead, viral spread into the subarachnoid space in rCDV(SH)-infected animals was triggered by infection of vascular endothelial cells and the hematogenous spread of virus-infected leukocytes from meningeal blood vessels into the subarachnoid space. This resulted in widespread infection of cells of the pia and arachnoid mater of the leptomeninges over large areas of the cerebral hemispheres. The ability to sensitively assess the in vivo spread of a neurovirulent strain of CDV provides a novel model system to study the mechanisms of virus spread into the CSF and the pathogenesis of acute viral meningitis.

  4. A set of enhanced green fluorescent protein concatemers for quantitative determination of nuclear localization signal strength.

    Science.gov (United States)

    Böhm, Jennifer; Thavaraja, Ramya; Giehler, Susanne; Nalaskowski, Marcus M

    2017-09-15

    Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. ERG protein expression over time

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Brasso, Klaus; Thomsen, Frederik Birkebæk

    2015-01-01

    AIMS: We evaluated the consistency in ERG protein expression from diagnostic specimens through rebiopsies to radical prostatectomies in patients with clinically localised prostate cancer to investigate the validity of ERG status in biopsies. METHODS: ERG expression was assessed by immunohistochem......AIMS: We evaluated the consistency in ERG protein expression from diagnostic specimens through rebiopsies to radical prostatectomies in patients with clinically localised prostate cancer to investigate the validity of ERG status in biopsies. METHODS: ERG expression was assessed...

  6. Matching faces with emotional expressions

    Directory of Open Access Journals (Sweden)

    Wenfeng eChen

    2011-08-01

    Full Text Available There is some evidence that faces with a happy expression are recognized better than faces with other expressions. However, little is known about whether this happy face advantage also applies to perceptual face matching, and whether similar differences exist among other expressions. Using a sequential matching paradigm, we systematically compared the effects of seven basic facial expressions on identity recognition. Identity matching was quickest when a pair of faces had an identical happy/sad/neutral expression, poorer when they had a fearful/surprise/angry expression, and poorest when they had a disgust expression. Faces with a happy/sad/fear/surprise expression were matched faster than those with an anger/disgust expression when the second face in a pair had a neutral expression. These results demonstrate that effects of facial expression on identity recognition are not limited to happy faces when a learned face is immediately tested. The results suggest different influences of expression in perceptual matching and long-term recognition memory.

  7. Expressing emotions in blogs

    DEFF Research Database (Denmark)

    Rodriguez-Hidalgo, Carmina Rodriguez-Hidalgo; Tan, Ed S.; Verlegh, Peeter

    2017-01-01

    Textual paralanguage cues (TPC) have been signaled as effective emotion transmitters online. Though several studies have investigated their properties and occurrence, there remains a gap concerning their communicative impact within specific psychological processes, such as the social sharing...... of emotion (SSE, Rimé, 2009). This study content-analyzed Live Journal blogposts for the occurrence of TPC in three phases of online SSE: initiation, feedback and repost. We compared these to TPC on a second type of emotional expression, emotional venting. Based on Social Information processing theory (SIP......, Walther, 1992), and on the Emotional Mimicry in Context (EMC, Hess & Fischer, 2013) framework, we study predictive relationships in TPC usage in our phased model of online SSE. Results showed that TPC prevailed in SSE blogposts and strongly dominated in emotional venting posts. TPC was more common...

  8. Natural Art, False Expressions

    Directory of Open Access Journals (Sweden)

    Oscar Hernando Nossa García

    2011-05-01

    Full Text Available In the documentary My Kid Could Paint That, directed by Bar-Lev, which deals with Marla Olmstead, the child prodigy of painting, several interviews with persons in the art world are conducted, among them an artist who uses a magnifying glass and the thinnest brushes to do his work. This man, although happy for the success of the child’s abstract paintings, saw in the whole spectacle a mockery of art, and stood firmly by her work. The girl’s father, also an artist, was accused of plagiarism. Cameras entered the child’s studio in order to prove that Marla was the real artist. Why should such relevance be given to authorship? What is the cause of the dispute between the expressive and the rational?

  9. The Expressive Organization

    DEFF Research Database (Denmark)

    This text challenges beliefs about organizational identity, reputation, and branding. It contains a wealth of new ideas for finding the elusive answers to questions troubling contemporary organizations. How does an organization create a strong reputation? What are the implications of corporate br...... students of management, business strategy, accounting, marketing, and communication studies; MBA students; Managers and consultants.......This text challenges beliefs about organizational identity, reputation, and branding. It contains a wealth of new ideas for finding the elusive answers to questions troubling contemporary organizations. How does an organization create a strong reputation? What are the implications of corporate...... branding on organizational structures and processes? How do organizations discover their identities? These are some of the vexing problems addressed in this book by a diverse international team of contributors. According to the authors, the future lies with "the expressive organization". Such organizations...

  10. Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko; Harushima, Yoshiaki; Fujisawa, Hironori; Mochizuki, Takako; Fujita, Masahiro; Ohyanagi, Hajime; Kurata, Nori

    2015-01-01

    Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue

  11. Facial Expressivity at 4 Months: A Context by Expression Analysis.

    Science.gov (United States)

    Bennett, David S; Bendersky, Margaret; Lewis, Michael

    2002-01-01

    The specificity predicted by differential emotions theory (DET) for early facial expressions in response to 5 different eliciting situations was studied in a sample of 4-month-old infants (n = 150). Infants were videotaped during tickle, sour taste, jack-in-the-box, arm restraint, and masked-stranger situations and their expressions were coded second by second. Infants showed a variety of facial expressions in each situation; however, more infants exhibited positive (joy and surprise) than negative expressions (anger, disgust, fear, and sadness) across all situations except sour taste. Consistent with DET-predicted specificity, joy expressions were the most common in response to tickling, and were less common in response to other situations. Surprise expressions were the most common in response to the jack-in-the-box, as predicted, but also were the most common in response to the arm restraint and masked-stranger situations, indicating a lack of specificity. No evidence of predicted specificity was found for anger, disgust, fear, and sadness expressions. Evidence of individual differences in expressivity within situations, as well as stability in the pattern across situations, underscores the need to examine both child and contextual factors in studying emotional development. The results provide little support for the DET postulate of situational specificity and suggest that a synthesis of differential emotions and dynamic systems theories of emotional expression should be considered.

  12. What emotion does the "facial expression of disgust" express?

    Science.gov (United States)

    Pochedly, Joseph T; Widen, Sherri C; Russell, James A

    2012-12-01

    The emotion attributed to the prototypical "facial expression of disgust" (a nose scrunch) depended on what facial expressions preceded it. In two studies, the majority of 120 children (5-14 years) and 135 adults (16-58 years) judged the nose scrunch as expressing disgust when the preceding set included an anger scowl, but as angry when the anger scowl was omitted. An even greater proportion of observers judged the nose scrunch as angry when the preceding set also included a facial expression of someone about to be sick. The emotion attributed to the nose scrunch therefore varies with experimental context. PsycINFO Database Record (c) 2012 APA, all rights reserved.

  13. Generational Differences of Emotional Expression

    Institute of Scientific and Technical Information of China (English)

    李学勇

    2014-01-01

    As a kind of subjective psychological activity, emotion can only be known and perceived by a certain expressive form. Varies as the different main bodies, difference of emotional expression can be reflected not only among individuals but between generations. The old conceals their emotions inside, the young express their emotions boldly, and the middle-aged are rational and deep in their expressions. Facing and understanding such differences is the premise and foundation of the con-struction of a harmonious relationship between different generations.

  14. Precise Analysis of String Expressions

    DEFF Research Database (Denmark)

    Christensen, Aske Simon; Møller, Anders; Schwartzbach, Michael Ignatieff

    2003-01-01

    We perform static analysis of Java programs to answer a simple question: which values may occur as results of string expressions? The answers are summarized for each expression by a regular language that is guaranteed to contain all possible values. We present several applications of this analysis...... are automatically produced. We present extensive benchmarks demonstrating that the analysis is efficient and produces results of useful precision......., including statically checking the syntax of dynamically generated expressions, such as SQL queries. Our analysis constructs flow graphs from class files and generates a context-free grammar with a nonterminal for each string expression. The language of this grammar is then widened into a regular language...

  15. Oracle Application Express 4 Recipes

    CERN Document Server

    Zehoo, Edmund

    2011-01-01

    Oracle Application Express 4 Recipes provides an example-based approach to learning Application Express - the ground-breaking, rapid application development platform included with every Oracle Database license. The recipes format is ideal for the quick-study who just wants a good example or two to kick start their thinking and get pointed in the right direction. The recipes cover the gamut of Application Express development. Author and Application Express expert Edmund Zehoo shows how to create data entry screens, visualize data in the form of reports and charts, implement validation and back-

  16. Expression modeling for expression-invariant face recognition

    NARCIS (Netherlands)

    Haar, F.B. Ter; Veltkamp, R.C.

    2010-01-01

    Morphable face models have proven to be an effective tool for 3D face modeling and face recognition, but the extension to 3D face scans with expressions is still a challenge. The two main difficulties are (1) how to build a new morphable face model that deals with expressions, and (2) how to fit

  17. Human papillomavirus gene expression

    International Nuclear Information System (INIS)

    Chow, L.T.; Hirochika, H.; Nasseri, M.; Stoler, M.H.; Wolinsky, S.M.; Chin, M.T.; Hirochika, R.; Arvan, D.S.; Broker, T.R.

    1987-01-01

    To determine the role of tissue differentiation on expression of each of the papillomavirus mRNA species identified by electron microscopy, the authors prepared exon-specific RNA probes that could distinguish the alternatively spliced mRNA species. Radioactively labeled single-stranded RNA probes were generated from a dual promoter vector system and individually hybridized to adjacent serial sections of formalin-fixed, paraffin-embedded biopsies of condylomata. Autoradiography showed that each of the message species had a characteristic tissue distribution and relative abundance. The authors have characterized a portion of the regulatory network of the HPVs by showing that the E2 ORF encodes a trans-acting enhancer-stimulating protein, as it does in BPV-1 (Spalholz et al. 1985). The HPV-11 enhancer was mapped to a 150-bp tract near the 3' end of the URR. Portions of this region are duplicated in some aggressive strains of HPV-6 (Boshart and zur Hausen 1986; Rando et al. 1986). To test the possible biological relevance of these duplications, they cloned tandem arrays of the enhancer and demonstrated, using a chloramphenicol acetyltransferase (CAT) assay, that they led to dramatically increased transcription proportional to copy number. Using the CAT assays, the authors found that the E2 proteins of several papillomavirus types can cross-stimulate the enhancers of most other types. This suggests that prior infection of a tissue with one papillomavirus type may provide a helper effect for superinfection and might account fo the HPV-6/HPV-16 coinfections in condylomata that they have observed

  18. Aging effects on intestinal homeostasis associated with expansion and dysfunction of intestinal epithelial stem cells.

    Science.gov (United States)

    Moorefield, Emily C; Andres, Sarah F; Blue, R Eric; Van Landeghem, Laurianne; Mah, Amanda T; Santoro, M Agostina; Ding, Shengli

    2017-08-29

    Intestinal epithelial stem cells (IESCs) are critical to maintain intestinal epithelial function and homeostasis. We tested the hypothesis that aging promotes IESC dysfunction using old (18-22 months) and young (2-4 month) Sox9-EGFP IESC reporter mice. Different levels of Sox9-EGFP permit analyses of active IESC (Sox9-EGFP Low ), activatable reserve IESC and enteroendocrine cells (Sox9-EGFP High ), Sox9-EGFP Sublow progenitors, and Sox9-EGFP Negative differentiated lineages. Crypt-villus morphology, cellular composition and apoptosis were measured by histology. IESC function was assessed by crypt culture, and proliferation by flow cytometry and histology. Main findings were confirmed in Lgr5-EGFP and Lgr5-LacZ mice. Aging-associated gene expression changes were analyzed by Fluidigm mRNA profiling. Crypts culture from old mice yielded fewer and less complex enteroids. Histology revealed increased villus height and Paneth cells per crypt in old mice. Old mice showed increased numbers and hyperproliferation of Sox9-EGFP Low IESC and Sox9-EGFP High cells. Cleaved caspase-3 staining demonstrated increased apoptotic cells in crypts and villi of old mice. Gene expression profiling revealed aging-associated changes in mRNAs associated with cell cycle, oxidative stress and apoptosis specifically in IESC. These findings provide new, direct evidence for aging associated IESC dysfunction, and define potential biomarkers and targets for translational studies to assess and maintain IESC function during aging.

  19. Cell model for the study of receptor and regulatory functions of human proHB-EGF

    Directory of Open Access Journals (Sweden)

    N. V. Korotkevych

    2014-08-01

    Full Text Available Developing of new models and approaches, particularly with fluorescent techniques, for investigation of intracellular transport of proHB-EGF and its ligand-receptor complexes is strongly required. In order to create a model for studying proHB-EGF functions the genetic construction pEGFP-N1-proHB-EGF, encoding proHB-EGF-EGFP which is fluorescent-labeled form of proHB-EGF with enhanced green fluorescent protein EGFP in the cytoplasmic terminus of the molecule, was obtained. Eukaryotic cells expressing fusion protein proHB-EGF-EGFP on the cell surface were obtained by transfection with pEGFP-N1-proHB-EGF. Expressed in the Vero cells proHB-EGF-EGFP could bind fluorescent derivative of nontoxic receptor-binding subunit B of diphtheria toxin mCherry-SubB. After stimulation of transfected cells with TPA (12-O-Tetradecanoylphorbol-13-acetate, proHB-EGF-EGFP formed a fluorescentl-labeled C-terminal fragment of the molecule – CTF-EGFP. Thus, the obtained genetic construction pEGFP-N1-proHB-EGF could be helpful in visualization of molecules proHB-EGF and CTF in cells, may open new possibilities for the studying of their functions, such as receptor function of proHB-EGF for diphtheria toxin, intracellular translocation of CTF and provide possibilities for natural proHB-EGF ligands search.

  20. A preclinical model for noninvasive imaging of hypoxia-induced gene expression; comparison with an exogenous marker of tumor hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Wen Bixiu; Burgman, Paul; Zanzonico, Pat; O' Donoghue, Joseph; Li, Gloria C.; Ling, C. Clifton [Memorial Sloan-Kettering Cancer Center, Department of Medical Physics, New York (United States); Cai Shangde; Finn, Ron [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York (United States); Serganova, Inna [Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York (United States); Blasberg, Ronald; Gelovani, Juri [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York (United States); Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York (United States)

    2004-11-01

    Hypoxia is associated with tumor aggressiveness and is an important cause of resistance to radiation therapy and chemotherapy. Assays of tumor hypoxia could provide selection tools for hypoxia-modifying treatments. The purpose of this study was to develop and characterize a rodent tumor model with a reporter gene construct that would be transactivated by the hypoxia-inducible molecular switch, i.e., the upregulation of HIF-1. The reporter gene construct is the herpes simplex virus 1-thymidine kinase (HSV1-tk) fused with the enhanced green fluorescent protein (eGFP) under the regulation of an artificial hypoxia-responsive enhancer/promoter. In this model, tumor hypoxia would up-regulate HIF-1, and through the hypoxia-responsive promoter transactivate the HSV1-tkeGFPfusion gene. The expression of this reporter gene can be assessed with the {sup 124}I-labeled reporter substrate 2'-fluoro-2'-deoxy-1-{beta}-d-arabinofuranosyl-5-iodouracil ({sup 124}I-FIAU), which is phosphorylated by the HSV1-tk enzyme and trapped in the hypoxic cells. Animal positron emission tomography (microPET) and phosphor plate imaging (PPI) were used in this study to visualize the trapped {sup 124}I-FIAU, providing a distribution of the hypoxia-induced molecular events. The distribution of {sup 124}I-FIAU was also compared with that of an exogenous hypoxic cell marker, {sup 18}F-fluoromisonidazole (FMISO). Our results showed that {sup 124}I-FIAU microPET imaging of the hypoxia-induced reporter gene expression is feasible, and that the intratumoral distributions of {sup 124}I-FIAU and {sup 18}F-FMISO are similar. In tumor sections, detailed radioactivity distributions were obtained with PPI which also showed similarity between {sup 124}I-FIAU and {sup 18}F-FMISO. This reporter system is sufficiently sensitive to detect hypoxia-induced transcriptional activation by noninvasive imaging and might provide a valuable tool in studying tumor hypoxia and in validating existing and future

  1. Differential Gene Expression and Aging

    Directory of Open Access Journals (Sweden)

    Laurent Seroude

    2002-01-01

    Full Text Available It has been established that an intricate program of gene expression controls progression through the different stages in development. The equally complex biological phenomenon known as aging is genetically determined and environmentally modulated. This review focuses on the genetic component of aging, with a special emphasis on differential gene expression. At least two genetic pathways regulating organism longevity act by modifying gene expression. Many genes are also subjected to age-dependent transcriptional regulation. Some age-related gene expression changes are prevented by caloric restriction, the most robust intervention that slows down the aging process. Manipulating the expression of some age-regulated genes can extend an organism's life span. Remarkably, the activity of many transcription regulatory elements is linked to physiological age as opposed to chronological age, indicating that orderly and tightly controlled regulatory pathways are active during aging.

  2. Measuring facial expression of emotion.

    Science.gov (United States)

    Wolf, Karsten

    2015-12-01

    Research into emotions has increased in recent decades, especially on the subject of recognition of emotions. However, studies of the facial expressions of emotion were compromised by technical problems with visible video analysis and electromyography in experimental settings. These have only recently been overcome. There have been new developments in the field of automated computerized facial recognition; allowing real-time identification of facial expression in social environments. This review addresses three approaches to measuring facial expression of emotion and describes their specific contributions to understanding emotion in the healthy population and in persons with mental illness. Despite recent progress, studies on human emotions have been hindered by the lack of consensus on an emotion theory suited to examining the dynamic aspects of emotion and its expression. Studying expression of emotion in patients with mental health conditions for diagnostic and therapeutic purposes will profit from theoretical and methodological progress.

  3. Homeobox gene expression in Brachiopoda

    DEFF Research Database (Denmark)

    Altenburger, Andreas; Martinez, Pedro; Wanninger, Andreas

    2011-01-01

    (ectoderm) specification with co-opted functions in notochord formation in chordates and left/right determination in ambulacrarians and vertebrates. The caudal ortholog, TtrCdx, is first expressed in the ectoderm of the gastrulating embryo in the posterior region of the blastopore. Its expression stays......The molecular control that underlies brachiopod ontogeny is largely unknown. In order to contribute to this issue we analyzed the expression pattern of two homeobox containing genes, Not and Cdx, during development of the rhynchonelliform (i.e., articulate) brachiopod Terebratalia transversa...... completion of larval development, which is marked by a three-lobed body with larval setae. Expression starts at gastrulation in two areas lateral to the blastopore and subsequently extends over the animal pole of the gastrula. With elongation of the gastrula, expression at the animal pole narrows to a small...

  4. A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Christian eBarbato

    2014-02-01

    Full Text Available Neurodegeneration associated with amyloid β (Aβ peptide accumulation, synaptic loss, and memory impairment are pathophysiological features of Alzheimer's disease (AD. Numerous microRNAs regulate amyloid precursor protein (APP expression and metabolism. We previously reported that miR-101 is a negative regulator of APP expression in cultured hippocampal neurons. In this study, a search for predicted APP metabolism-associated miR-101 targets led to the identification of a conserved miR-101 binding site within the 3’ untranslated region (UTR of the mRNA encoding Ran-binding protein 9 (RanBP9. RanBP9 increases APP processing by β-amyloid converting enzyme 1 (BACE1, secretion of soluble APPβ (sAPPβ, and generation of Aβ. MiR-101 significantly reduced reporter gene expression when co-transfected with a RanBP9 3'-UTR reporter construct, while site-directed mutagenesis of the predicted miR-101 target site eliminated the reporter response. To investigate the effect of stable inhibition of miR-101 both in vitro and in vivo, a microRNA sponge was developed to bind miR-101 and derepress its targets. Four tandem bulged miR-101 responsive elements (REs, located downstream of the enhanced green fluorescence protein (EGFP open reading frame and driven by the synapsin promoter, were placed in a lentiviral vector to create the pLSyn-miR-101 sponge. Delivery of the sponge to primary hippocampal neurons significantly increased both APP and RanBP9 expression, as well as sAPPβ levels in the conditioned medium. Importantly, silencing of endogenous RanBP9 reduced sAPPβ levels in miR-101 sponge-containing hippocampal cultures, indicating that miR-101 inhibition may increase amyloidogenic processing of APP by RanBP9. Lastly, the impact of miR-101 on its targets was demonstrated in vivo by intrahippocampal injection of the pLSyn-miR-101 sponge into C57BL6 mice. This study thus provides the basis for studying the consequences of long-term miR-101 inhibition on

  5. Nestin expression in neuroepithelial tumors.

    Science.gov (United States)

    Schiffer, Davide; Manazza, Andrea; Tamagno, Ilaria

    2006-05-29

    Nestin is a marker of early stages of neurocytogenesis. It has been studied in 50 neuroepithelial tumors, mostly gliomas of different malignancy grades, by immunohistochemistry, immunofluorescence, immunoblotting, and confocal microscopy and compared with GFAP and Vimentin. As an early marker of differentiation, Nestin is almost not expressed in diffuse astrocytomas, variably expressed in anaplastic astrocytomas and strongly and irregularly expressed in glioblastomas. Negative in oligodendrogliomas, it stains ependymomas and shows a gradient of expression in pilocytic astrocytomas. In glioblastomas, Nestin distribution does not completely correspond to that of GFAP and Vimentin with which its expression varies in tumor cells in a complementary way, as confirmed by confocal microscopy. Tumor cells can thus either derive from or differentiate toward the neurocytogenetic stages. Hypothetically, they could be put in relation with radial glia where during embriogenesis the three antigens are successively expressed. Completely negative cells of invasive or recurrent glioblastomas may represent malignant selected clones after accumulation of mutations or early stem cells not expressing antigens.

  6. The expressive stance: intentionality, expression, and machine art

    OpenAIRE

    Linson, Adam

    2013-01-01

    This paper proposes a new interpretive stance for interpreting artistic works and performances that is relevant to artificial intelligence research but also has broader implications. Termed the expressive stance, this stance makes intelligible a critical distinction between present-day machine art and human art, but allows for the possibility that future machine art could find a place alongside our own. The expressive stance is elaborated as a response to Daniel Dennett's notion of the intent...

  7. [Prosopagnosia and facial expression recognition].

    Science.gov (United States)

    Koyama, Shinichi

    2014-04-01

    This paper reviews clinical neuropsychological studies that have indicated that the recognition of a person's identity and the recognition of facial expressions are processed by different cortical and subcortical areas of the brain. The fusiform gyrus, especially the right fusiform gyrus, plays an important role in the recognition of identity. The superior temporal sulcus, amygdala, and medial frontal cortex play important roles in facial-expression recognition. Both facial recognition and facial-expression recognition are highly intellectual processes that involve several regions of the brain.

  8. Expression in E. coli systems

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Kristiansen, Karsten; Nøhr, Jane

    2003-01-01

    intracellularly in soluble form. In E. coli, proteins containing disulfide bonds are best produced by secretion because the disulfide forming foldases reside in the periplasm. Likewise, a correct N-terminus is more likely to be obtained upon secretion. Moreover, potentially toxic proteins are more likely......Owing to cost advantage, speed of production, and often high product yield (up to 50% of total cell protein), expression in Escherichia coli is generally the first choice when attempting to express a recombinant protein. Expression systems exist to produce recombinant protein intracellularly...

  9. Analysis of Facial Expression by Taste Stimulation

    Science.gov (United States)

    Tobitani, Kensuke; Kato, Kunihito; Yamamoto, Kazuhiko

    In this study, we focused on the basic taste stimulation for the analysis of real facial expressions. We considered that the expressions caused by taste stimulation were unaffected by individuality or emotion, that is, such expressions were involuntary. We analyzed the movement of facial muscles by taste stimulation and compared real expressions with artificial expressions. From the result, we identified an obvious difference between real and artificial expressions. Thus, our method would be a new approach for facial expression recognition.

  10. A Tattoo Is Expression, Too.

    Science.gov (United States)

    Dowling-Sendor, Benjamin

    1997-01-01

    In "Stephenson v. Davenport Community School District," the U.S. Eighth Circuit Court of Appeals ruled that schools cannot adopt unduly vague policies to regulate student expression, in this case, a cross-shaped tattoo. (LMI)

  11. Online handwritten mathematical expression recognition

    Science.gov (United States)

    Büyükbayrak, Hakan; Yanikoglu, Berrin; Erçil, Aytül

    2007-01-01

    We describe a system for recognizing online, handwritten mathematical expressions. The system is designed with a user-interface for writing scientific articles, supporting the recognition of basic mathematical expressions as well as integrals, summations, matrices etc. A feed-forward neural network recognizes symbols which are assumed to be single-stroke and a recursive algorithm parses the expression by combining neural network output and the structure of the expression. Preliminary results show that writer-dependent recognition rates are very high (99.8%) while writer-independent symbol recognition rates are lower (75%). The interface associated with the proposed system integrates the built-in recognition capabilities of the Microsoft's Tablet PC API for recognizing textual input and supports conversion of hand-drawn figures into PNG format. This enables the user to enter text, mathematics and draw figures in a single interface. After recognition, all output is combined into one LATEX code and compiled into a PDF file.

  12. Emerald Express '95: Analysis Report

    National Research Council Canada - National Science Library

    Newett, Sandra

    1996-01-01

    The purpose of Emerald Express was to bring together senior representatives from military, relief, political, and diplomatic communities to address issues that arise during Humanitarian Assistance and Peace Operations (HA/POs...

  13. Race, Reparations, and Free Expression.

    Science.gov (United States)

    Brownstein, Andrew

    2001-01-01

    Describes how a controversial newspaper ad opposing slavery reparations and the subsequent trashing of the student daily have set off a debate at Brown University about the competing values of sensitivity and free expression. (EV)

  14. Expression of Recombinant Human Butyrylcholinesterase

    National Research Council Canada - National Science Library

    Lockridge, Oksana

    1997-01-01

    .... The G117H enzyme has the potential to be useful for decontamination of skin and eye. To determine how many amino acids could be deleted from butyrylcholinesterase without loss of activity, deletion mutants were expressed...

  15. Regulation of eucaryotic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Brent, R.; Ptashne, M.S

    1989-05-23

    This patent describes a method of regulating the expression of a gene in a eucaryotic cell. The method consists of: providing in the eucaryotic cell, a peptide, derived from or substantially similar to a peptide of a procaryotic cell able to bind to DNA upstream from or within the gene, the amount of the peptide being sufficient to bind to the gene and thereby control expression of the gene.

  16. A Herpes Simplex Virus Type 1 Mutant Expressing a Baculovirus Inhibitor of Apoptosis Gene in Place of Latency-Associated Transcript Has a Wild-Type Reactivation Phenotype in the Mouse

    Science.gov (United States)

    Jin, Ling; Perng, Guey-Chuen; Mott, Kevin R.; Osorio, Nelson; Naito, Julia; Brick, David J.; Carpenter, Dale; Jones, Clinton; Wechsler, Steven L.

    2005-01-01

    The latency-associated transcript (LAT) is essential for the wild-type herpes simplex virus type 1 (HSV-1) high-reactivation phenotype since LAT− mutants have a low-reactivation phenotype. We previously reported that LAT can decrease apoptosis and proposed that this activity is involved in LAT's ability to enhance the HSV-1 reactivation phenotype. The first 20% of the primary 8.3-kb LAT transcript is sufficient for enhancing the reactivation phenotype and for decreasing apoptosis, supporting this proposal. For this study, we constructed an HSV-1 LAT− mutant that expresses the baculovirus antiapoptosis gene product cpIAP under control of the LAT promoter and in place of the LAT region mentioned above. Mice were ocularly infected with this mutant, designated dLAT-cpIAP, and the reactivation phenotype was determined using the trigeminal ganglion explant model. dLAT-cpIAP had a reactivation phenotype similar to that of wild-type virus and significantly higher than that of (i) the LAT− mutant dLAT2903; (ii) dLAT1.5, a control virus containing the same LAT deletion as dLAT-cpIAP, but with no insertion of foreign DNA, thereby controlling for potential readthrough transcription past the cpIAP insert; and (iii) dLAT-EGFP, a control virus identical to dLAT-cpIAP except that it contained the enhanced green fluorescent protein open reading frame (ORF) in place of the cpIAP ORF, thereby controlling for expression of a random foreign gene instead of the cpIAP gene. These results show that an antiapoptosis gene with no sequence similarity to LAT can efficiently substitute for the LAT function involved in enhancing the in vitro-induced HSV-1 reactivation phenotype in the mouse. PMID:16160155

  17. Expression of FLNa in human melanoma cells regulates the function of integrin α1β1 and phosphorylation and localisation of PKB/AKT/ERK1/2 kinases.

    Science.gov (United States)

    Krebs, Kristi; Ruusmann, Anu; Simonlatser, Grethel; Velling, Teet

    2015-12-01

    FLNa is a ubiquitous cytoskeletal protein that links transmembrane receptors, including integrins, to F-actin and functions as a signalling intermediate. We investigated FLNa's role in the function of integrin-type collagen receptors, EGF-EGFR signalling and regulation of PKB/Akt and ERK1/2. Using FLNa-deficient M2 human melanoma cells, and same cells expressing EGFP-FLNa (M2F) or its Ig-like repeats 1-8+24, 8-15+24 and 16-24, we found that in M2F and M2 8-15+24 cells, EGF induced the increased phosphorylation of PKB/Akt and ERK1/2. In M2F cells EGF induced the localisation of these kinases to cell nucleus and lamellipodia, respectively, and the ERK1/2 phosphorylation-dependent co-immunoprecipitation of FLNa with ERK1/2. Only M2F and M2 8-15+24 cells adhered to and spread on type I collagen whereas on fibronectin all cells behaved similarly. α1β1 and α2β1 were the integrin-type collagen receptors expressed on these cells with primarily α1β1 localising to focal contacts and affecting cell adhesion and migration in a manner dependent on FLNa or its Ig-like repeats 8-15. Our results suggest a role for FLNa repeats 8-15 in the α1-subunit-dependent regulation of integrin α1β1 function, EGF-EGFR signalling to PKB/Akt and ERK1/2, identify ERK1/2 in EGF-induced FLNa-associated protein complexes, and show that the function of different integrins is subjected to differential regulation by FLNa. Copyright © 2015. Published by Elsevier GmbH.

  18. Craniopharyngioma: Survivin expression and ultrastructure

    Science.gov (United States)

    ZHU, JIANG; YOU, CHAO

    2015-01-01

    The aim of the present study was to investigate the significance of survivin protein expression levels in craniopharyngioma. Tumor samples and clinical data were obtained from 50 patients with craniopharyngioma who were admitted to the West China Hospital of Sichuan University (Chengdu, China). The morphology of the craniopharyngioma samples was observed using optical and electron microscopes, and survivin expression was investigated in the samples by immunohistochemical analysis. The immunohistochemical results revealed survivin expression in all of the craniopharyngioma samples, but not in the healthy brain tissue samples. It was identified that survivin was expressed at a higher level in cases of the adamantinomatous type compared with those of the squamous-papillary type, in male patients compared with female patients, in children compared with adults and in recurrent cases compared with non-recurrent cases. Furthermore, no significant difference was detected in survivin expression levels among the tumors of different subtypes and different disease stages. The results of the present study indicate that survivin is significant in the development of craniopharyngioma, and that survivin protein expression levels are a meaningful indicator for assessing craniopharyngioma recurrence. PMID:25435936

  19. The motivation to express prejudice

    Science.gov (United States)

    Forscher, Patrick S.; Cox, William T. L.; Graetz, Nicholas; Devine, Patricia G.

    2015-01-01

    Contemporary prejudice research focuses primarily on people who are motivated to respond without prejudice and the ways in which unintentional bias can cause these people to act inconsistent with this motivation. However, some real-world phenomena (e.g., hate speech, hate crimes) and experimental findings (e.g., Plant & Devine, 2001; 2009) suggest that some expressions of prejudice are intentional. These phenomena and findings are difficult to explain solely from the motivations to respond without prejudice. We argue that some people are motivated to express prejudice, and we develop the motivation to express prejudice (MP) scale to measure this motivation. In seven studies involving more than 6,000 participants, we demonstrate that, across scale versions targeted at Black people and gay men, the MP scale has good reliability and convergent, discriminant, and predictive validity. In normative climates that prohibit prejudice, the internal and external motivations to express prejudice are functionally non-independent, but they become more independent when normative climates permit more prejudice toward a target group. People high in the motivation to express prejudice are relatively likely to resist pressure to support programs promoting intergroup contact and vote for political candidates who support oppressive policies. The motivation to express prejudice predicted these outcomes even when controlling for attitudes and the motivations to respond without prejudice. This work encourages contemporary prejudice researchers to broaden the range of samples, target groups, and phenomena that they study, and more generally to consider the intentional aspects of negative intergroup behavior. PMID:26479365

  20. Pericentrin expression in Down's syndrome.

    Science.gov (United States)

    Salemi, Michele; Barone, Concetta; Romano, Carmelo; Salluzzo, Roberto; Caraci, Filippo; Cantarella, Rita Anna; Salluzzo, Maria Grazia; Drago, Filippo; Romano, Corrado; Bosco, Paolo

    2013-11-01

    Down's syndrome (DS) is the most frequent genetic cause of intellectual disability and is a chromosomal abnormality of chromosome 21 trisomy. The pericentrin gene (PCNT) has sequenced in 21q22.3 inside of the minimal critical region for Down's syndrome. Alterations of PCNT gene are associated with dwarfism, cardiomyopathy and other pathologies. In this study, we have evaluated the possible differential expression of PCNT mRNA, by qRT-PCR, in peripheral blood leukocytes of DS subjects compared with the normal population. In the present case-control study, PCNT gene expression was increased by 72.72% in 16 out 22 DS samples compared with normal subjects. Our data suggest that changes in the expression levels of PCNT in DS subjects may be involved into the molecular mechanism of Down's syndrome.

  1. Converting nested algebra expressions into flat algebra expressions

    NARCIS (Netherlands)

    Paredaens, J.; Van Gucht, D.

    1992-01-01

    Nested relations generalize ordinary flat relations by allowing tuple values to be either atomic or set valued. The nested algebra is a generalization of the flat relational algebra to manipulate nested relations. In this paper we study the expressive power of the nested algebra relative to its

  2. EXPRESS Service to the International Space Station: EXPRESS Pallet

    Science.gov (United States)

    Primm, Lowell; Bergmann, Alan

    1998-01-01

    The International Space Station (ISS) will be the ultimate scientific accomplishment in the history of NASA, with its primary objective of providing unique scientific investigation opportunities. This objective is the basis for the creation of the EXPRESS Pallet System (ExPs). The EXPRESS Pallet will provide extremal/unpressurized accommodations for a wide variety of external users. The payload developers represent many science disciplines, including earth observation, communications, solar and deep space viewing, long-term exposure, and many others. The EXPRESS Pallet will provide a mechanism to maximum utilization of the limited ISS unpressurized payload volume, standard physical payload interfaces for users, a standard integration template for users and the capability to changeout payloads on-orbit. The EXPRESS Pallet provides access to Ram, Wake, Starboard, Port, Nadir, Zenith and Earth Limb for exposure and viewing. 'Me ExPs consists of the Pallet structure, payload Adapters, and a subsystem assembly which includes data controller, power distribution and conversion, and Extra Vehicular Robotics/Extra-Vehicular Activity systems.

  3. Construction of PVX virus-expression vector to express enterotoxin ...

    African Journals Online (AJOL)

    Potato X potyvirus (PVX)-based vector has been comprehensively applied in transient expression system. In order to produce the heterologous proteins more quickly and stably, the ClaI and NotI enzyme sites were introduced into the Enterotoxin fusion gene LTB-ST by polymerase chain reaction (PCR) and the LTB-ST ...

  4. Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

    International Nuclear Information System (INIS)

    Hayashi, Masami; Shimada, Yukiko; Inomata, Mitsushi; Ohno-Iwashita, Yoshiko

    2006-01-01

    The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with β-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than to intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

  5. Advanced express web application development

    CERN Document Server

    Keig, Andrew

    2013-01-01

    A practical book, guiding the reader through the development of a single page application using a feature-driven approach.If you are an experienced JavaScript developer who wants to build highly scalable, real-world applications using Express, this book is ideal for you. This book is an advanced title and assumes that the reader has some experience with node, Javascript MVC web development frameworks, and has heard of Express before, or is familiar with it. You should also have a basic understanding of Redis and MongoDB. This book is not a tutorial on Node, but aims to explore some of the more

  6. Gene expression in colorectal cancer

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Christensen, Lise Lotte; Olesen, Sanne Harder

    2002-01-01

    Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each...... pool) of total RNA from left-sided sporadic colorectal carcinomas. We compared normal tissue to carcinoma tissue from Dukes' stages A-D (noninvasive to distant metastasis) and identified 908 known genes and 4,155 ESTs that changed remarkably from normal to tumor tissue. Based on intensive filtering 226...

  7. Correction of gene expression data

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Stewart, C. Neal, Jr.; Noeparvar, Shahin

    2014-01-01

    This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies....... For maximal reliability of analysis, therefore, comparisons should be performed at the cellular level. This could be accomplished using an appropriate correction method that can detect and remove the inter-treatment bias for cell-number. Based on inter-treatment variations of reference genes, we introduce...

  8. Microsoft Expression Web for dummies

    CERN Document Server

    Hefferman, Linda

    2013-01-01

    Expression Web is Microsoft's newest tool for creating and maintaining dynamic Web sites. This FrontPage replacement offers all the simple ""what-you-see-is-what-you-get"" tools for creating a Web site along with some pumped up new features for working with Cascading Style Sheets and other design options. Microsoft Expression Web For Dummies arrives in time for early adopters to get a feel for how to build an attractive Web site. Author Linda Hefferman teams up with longtime FrontPage For Dummies author Asha Dornfest to show the easy way for first-time Web designers, FrontPage ve

  9. The change of expression configuration affects identity-dependent expression aftereffect but not identity-independent expression aftereffect

    Directory of Open Access Journals (Sweden)

    Miao eSong

    2015-12-01

    Full Text Available The present study examined the influence of expression configuration on cross-identity expression aftereffect. The expression configuration refers to the spatial arrangement of facial features in a face for conveying an emotion, e.g., an open-mouth smile versus a closed-mouth smile. In the first of two experiments, the expression aftereffect is measured using across-identity/cross-expression configuration factorial design. The facial identities of test faces were the same or different from the adaptor, while orthogonally, the expression configurations of those facial identities were also the same or different. The result shows that the change of expression configuration impaired the expression aftereffect when the facial identities of adaptor and tests were the same; however, the impairment effect disappears when facial identities were different, indicating the identity-independent expression representation is more robust to the change of the expression configuration in comparison with the identity-dependent expression representation. In the second experiment, we used schematic line faces as adaptors and real faces as tests to minimize the similarity between the adaptor and tests, which is expected to exclude the contribution from the identity-dependent expression representation to expression aftereffect. The second experiment yields a similar result as the identity-independent expression aftereffect observed in Experiment 1. The findings indicate the different neural sensitivities to expression configuration for identity-dependent and identity-independent expression systems.

  10. 'Endurance' Courtesy of Mars Express

    Science.gov (United States)

    2004-01-01

    NASA's Mars Exploration Rover Opportunity used its panoramic camera to capture this false-color image of the interior of 'Endurance Crater' on the rover's 188th martian day (Aug. 4, 2004). The image data were relayed to Earth by the European Space Agency's Mars Express orbiter. The image was generated from separate frames using the cameras 750-, 530- and 480-nanometer filters.

  11. Communicating emotions in expressive avatars

    NARCIS (Netherlands)

    Kierkegaard, P.M.

    2010-01-01

    Avatars have become a fundamental part of collaborative virtual environments. They are the visual embodiment of the user and are designed to address key issues in the interaction process between the user and the CVE. Giving avatars expressive abilities has been considered essential in computer-human

  12. Spirit Boxes: Expressions of Culture.

    Science.gov (United States)

    DeMuro, Ted

    1984-01-01

    After studying the culture and art of the ancient civilizations of South America, Mesopotamia, Greece, and Egypt, secondary level art students made spirit boxes as expressions of the various cultures. How to make the boxes and how to prepare the face molds are described. (RM)

  13. Mapping and Manipulating Facial Expression

    Science.gov (United States)

    Theobald, Barry-John; Matthews, Iain; Mangini, Michael; Spies, Jeffrey R.; Brick, Timothy R.; Cohn, Jeffrey F.; Boker, Steven M.

    2009-01-01

    Nonverbal visual cues accompany speech to supplement the meaning of spoken words, signify emotional state, indicate position in discourse, and provide back-channel feedback. This visual information includes head movements, facial expressions and body gestures. In this article we describe techniques for manipulating both verbal and nonverbal facial…

  14. The SSETI-express Mission

    DEFF Research Database (Denmark)

    Alminde, Lars; Bisgaard, Morten; Melville, N.

    In January 2004 a group of students met at the European Space Technology and Research Centre (ESTEC) in Holland to discuss the feasibility of building a micro-satellite, dubbed SSETI-Express, from parts derived from other student satellite projects and launch it within one and a half year...

  15. The SSETI-Express Mission

    DEFF Research Database (Denmark)

    Alminde, Lars; Bisgaard, Morten; Melville, Neil

    2005-01-01

    In January 2004 a group of students met at the European Space Technology and Research Centre (ESTEC) in Holland to discuss the feasibility of building a micro-satellite, dubbed SSETI-Express, from parts derived from other student satellite projects and launch it within one and a half year...

  16. The motivation to express prejudice.

    Science.gov (United States)

    Forscher, Patrick S; Cox, William T L; Graetz, Nicholas; Devine, Patricia G

    2015-11-01

    Contemporary prejudice research focuses primarily on people who are motivated to respond without prejudice and the ways in which unintentional bias can cause these people to act in a manner inconsistent with this motivation. However, some real-world phenomena (e.g., hate speech, hate crimes) and experimental findings (e.g., Plant & Devine, 2001, 2009) suggest that some prejudice is intentional. These phenomena and findings are difficult to explain solely from the motivations to respond without prejudice. We argue that some people are motivated to express prejudice, and we develop the Motivation to Express Prejudice Scale (MP) to measure this motivation. In 7 studies involving more than 6,000 participants, we demonstrate that, across scale versions targeted at Black people and gay men, the MP has good reliability and convergent, discriminant, and predictive validity. In normative climates that prohibit prejudice, the internal and external motivations to express prejudice are functionally nonindependent, but they become more independent when normative climates permit more prejudice toward a target group. People high in the motivation to express prejudice are relatively likely to resist pressure to support programs promoting intergroup contact and to vote for political candidates who support oppressive policies. The motivation to express prejudice predicted these outcomes even when controlling for attitudes and the motivations to respond without prejudice. This work encourages contemporary prejudice researchers to give greater consideration to the intentional aspects of negative intergroup behavior and to broaden the range of phenomena, target groups, and samples that they study. (c) 2015 APA, all rights reserved).

  17. Vascular Gene Expression: A Hypothesis

    Directory of Open Access Journals (Sweden)

    Angélica Concepción eMartínez-Navarro

    2013-07-01

    Full Text Available The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular tissues. This tissue-specific expression in Arabidopsis is predicted by the overrepresentation of GA/CT-rich motifs in gene promoters. In this work we have searched for common motifs in upstream regions of the homologous genes from plants considered to possess a primitive vascular tissue (a lycophyte, as well as from others that lack a true vascular tissue (a bryophyte, and finally from chlorophytes. Both lycophyte and bryophyte display motifs similar to those found in Arabidopsis with a significantly low E-value, while the chlorophytes showed either a different conserved motif or no conserved motif at all. These results suggest that these same genes are expressed coordinately in non- vascular plants; this coordinate expression may have been one of the prerequisites for the development of conducting tissues in plants. We have also analyzed the phylogeny of conserved proteins that may be involved in phloem function and development. The presence of CmPP16, APL, FT and YDA in chlorophytes suggests the recruitment of ancient regulatory networks for the development of the vascular tissue during evolution while OPS is a novel protein specific to vascular plants.

  18. DNA expressions - A formal notation for DNA

    NARCIS (Netherlands)

    Vliet, Rudy van

    2015-01-01

    We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

  19. Recombination-ready Sindbis replicon expression vectors for transgene expression

    Directory of Open Access Journals (Sweden)

    Olson Ken E

    2007-10-01

    Full Text Available Abstract Background Sindbis viruses have been widely used as tools to study gene function in cells. Despite the utility of these systems, the construction and production of alphavirus replicons is time consuming and inefficient due to potential additional restriction sites within the insert region and lack of directionality for insert ligation. In this report, we present a system useful for producing recombinant Sindbis replicons that uses lambda phage recombination technology to rapidly and specifically construct replicon expression plasmids that contain insert regions in the desired orientation. Results Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert. Replicons were easily produced in cell culture and packaged into pseudo-infectious viral particles. Insect and mammalian cells infected with pseudo-infectious viral particles expressed various transgenes at high levels. Finally, inserts from persistently replicating replicon RNA were easily isolated and recombined back into entry plasmids for sequencing and subsequent analysis. Conclusion Replication-ready replicon expression plasmids make the use of alphavirus replicons fast and easy as compared to traditional replicon production methods. This system represents a significant step forward in the utility and ease of use of alphavirus replicons in the study of gene function.

  20. Dynamic Facial Expression of Emotion Made Easy

    OpenAIRE

    Broekens, Joost; Qu, Chao; Brinkman, Willem-Paul

    2012-01-01

    Facial emotion expression for virtual characters is used in a wide variety of areas. Often, the primary reason to use emotion expression is not to study emotion expression generation per se, but to use emotion expression in an application or research project. What is then needed is an easy to use and flexible, but also validated mechanism to do so. In this report we present such a mechanism. It enables developers to build virtual characters with dynamic affective facial expressions. The mecha...

  1. Nestin Reporter Transgene Labels Multiple Central Nervous System Precursor Cells

    Directory of Open Access Journals (Sweden)

    Avery S. Walker

    2010-01-01

    Full Text Available Embryonic neuroepithelia and adult subventricular zone (SVZ stem and progenitor cells express nestin. We characterized a transgenic line that expresses enhanced green fluorescent protein (eGFP specified to neural tissue by the second intronic enhancer of the nestin promoter that had several novel features. During embryogenesis, the dorsal telencephalon contained many and the ventral telencephalon few eGFP+ cells. eGFP+ cells were found in postnatal and adult neurogenic regions. eGFP+ cells in the SVZ expressed multiple phenotype markers, glial fibrillary acidic protein, Dlx, and neuroblast-specific molecules suggesting the transgene is expressed through the lineage. eGFP+ cell numbers increased in the SVZ after cortical injury, suggesting this line will be useful in probing postinjury neurogenesis. In non-neurogenic regions, eGFP was strongly expressed in oligodendrocyte progenitors, but not in astrocytes, even when they were reactive. This eGFP+ mouse will facilitate studies of proliferative neuroepithelia and adult neurogenesis, as well as of parenchymal oligodendrocytes.

  2. IL-33 activates eosinophils of visceral adipose tissue both directly and via innate lymphoid cells.

    Science.gov (United States)

    Hashiguchi, Masaaki; Kashiwakura, Yuji; Kojima, Hidefumi; Kobayashi, Ayano; Kanno, Yumiko; Kobata, Tetsuji

    2015-03-01

    Eosinophils are multifunctional leukocytes involved in allergic reactions as well as adipose tissue regulation. IL-5 is required for eosinophil survival; however, the in vivo mechanisms of eosinophil regulation are not fully understood. A tg mouse model with il5 promoter-driven EGFP expression was established for detecting the IL-5-producing cells in vivo. Il5-egfp tg mice expressed high levels of EGFP in gonadal adipose tissue (GAT) cells. EGFP(+) cells in GAT were mainly group 2 innate lymphoid cells (ILCs). IL-33 preferentially expanded EGFP(+) cells and eosinophils in GAT in vivo. EGFP(+) ILCs were found to upregulate prg2 mRNA expression in GAT eosinophils. These results demonstrate that ILCs activate eosinophils in GAT. The blockage of IL-33Rα, on the other hand, did not impair EGFP(+) ILC numbers but did impair eosinophil numbers in vivo. GAT eosinophils expressed IL-33Rα and IL-33 expanded eosinophil numbers in CD90(+) cell-depleted mice. IL-33 was further observed to induce the expression of retnla and epx mRNA in eosinophils. These findings demonstrate that IL-33 directly activates eosinophils in GAT, and together with our other findings described above, our findings show that IL-33 has dual pathways via which it activates eosinophils in vivo: a direct activation pathway and a group 2 ILC-mediated pathway. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Emotional Expression in Reality TV

    DEFF Research Database (Denmark)

    Rasmussen, Tove Arendt

    Reality TV has been disputed and discussed along many lines for the last 20 years; the media themselves always with a twist of concern about reality programs crossing moral borderlines while research has put more interest in questions of genre and representation of more or less useful and maybe...... empowering social identities. In the latest generation of reality TV which Misha Kava has described as the “celebrity generation”, it seems that reality creates new standards for mixing facts and fiction and even different “realities” as participants become media stars while more traditional celebrities...... are being treated as ordinary people. My article will discuss different presentations of selves and especially the emotional verbal and nonverbal expressions in reality TV communication. Aspects of the intimate self and its emotional expressions seem to be strategically managed in reality TV and even...

  4. On the Expressiveness of Joining

    Directory of Open Access Journals (Sweden)

    Thomas Given-Wilson

    2015-08-01

    Full Text Available The expressiveness of communication primitives has been explored in a common framework based on the pi-calculus by considering four features: synchronism (asynchronous vs synchronous, arity (monadic vs polyadic data, communication medium (shared dataspaces vs channel-based, and pattern-matching (binding to a name vs testing name equality vs intensionality. Here another dimension coordination is considered that accounts for the number of processes required for an interaction to occur. Coordination generalises binary languages such as pi-calculus to joining languages that combine inputs such as the Join Calculus and general rendezvous calculus. By means of possibility/impossibility of encodings, this paper shows coordination is unrelated to the other features. That is, joining languages are more expressive than binary languages, and no combination of the other features can encode a joining language into a binary language. Further, joining is not able to encode any of the other features unless they could be encoded otherwise.

  5. Architecture between construction and expression

    Directory of Open Access Journals (Sweden)

    Luca Lanini

    2015-11-01

    Full Text Available I think that the problem is basically the same as always: to redefine the relationship between Construction and Expression. On one hand, construction is considered an endeavor, on the other hand, a tendency to contest architecture only with expression. The relationship between architecture and control of urban phenomena is a relationship that has been gradually lost in the past three decades and has been replaced by the relationship between finance and planning. A useful object is very different from a building: for its time and costs of construction. Buildings are expected to last many years and are paid with everybody’s taxes, a design object’s lifespan is perhaps fifty years, then it’s ready to be modernized or for the landfill ... I believe that the main tool that a designer must have, beyond necessary technical skills, is the capacity of his/her own critical thought.

  6. Transgenic Arabidopsis Gene Expression System

    Science.gov (United States)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  7. Podoplanin Expression in Canine Melanoma

    OpenAIRE

    Ogasawara, Satoshi; Honma, Ryusuke; Kaneko, Mika K.; Fujii, Yuki; Kagawa, Yumiko; Konnai, Satoru; Kato, Yukinari

    2016-01-01

    A type I transmembrane protein, podoplanin (PDPN), is expressed in several normal cells such as lymphatic endothelial cells or pulmonary type I alveolar cells. We recently demonstrated that anticanine PDPN monoclonal antibody (mAb), PMab-38, recognizes canine PDPN of squamous cell carcinomas, but does not react with lymphatic endothelial cells. Herein, we investigated whether PMab-38 reacts with canine melanoma. PMab-38 reacted with 90% of melanoma cells (9/10 cases) using immunohistochemistr...

  8. Summer Oral Expression English Course

    CERN Document Server

    HR Department

    2011-01-01

    An English Oral Expression course will take place between 15 August and 30 September 2011. Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enrol here. Or contact: Kerstin FUHRMEISTER (70896) Tessa OSBORNE (72957)  

  9. Children's expression through play therapy

    Directory of Open Access Journals (Sweden)

    Ljubomirović Nataša

    2015-01-01

    Full Text Available Play as a child's expression, is a skill through which children speaks to adults. Play therapy is a broad field of therapeutic intervention based on the play in order to help the child to cope with problems. Through play, children learn to communicate with others, to express their feelings. Through play they learn and can improve their cognitive, emotional and social capabilities. Play therapy is a nondirective technique focused on the child. It is not focused on the problem, at present even the past, but focused on the expression of the child feelings, accepting the child, rather than correction. The focus has been on the wisdom of a child, not on expertise therapists, guiding the child through play rather than instructing. The aim of play therapy is to encourage healthy growth and development, developing skills in problem solving, reduction of undesirable behavior, confidence building and the development of self-control. This method is effective for a wide range of children's problems, such as the state of stress, anxiety, problem behavior, hyperkinetic syndrome, depression, loss, trauma, the problem of bonding situations parents divorced, somatic disorders, autism spectrum disorders, social problems.

  10. Gene expression profile of pulpitis.

    Science.gov (United States)

    Galicia, J C; Henson, B R; Parker, J S; Khan, A A

    2016-06-01

    The cost, prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis. This study was aimed to identify the genetic contributors to pulpal pain and inflammation. Inflamed pulps were collected from patients diagnosed with irreversible pulpitis (n=20). Normal pulps from teeth extracted for various reasons served as controls (n=20). Pain level was assessed using a visual analog scale (VAS). Genome-wide microarray analysis was performed using Affymetrix GeneTitan Multichannel Instrument. The difference in gene expression levels were determined by the significance analysis of microarray program using a false discovery rate (q-value) of 5%. Genes involved in immune response, cytokine-cytokine receptor interaction and signaling, integrin cell surface interactions, and others were expressed at relatively higher levels in the pulpitis group. Moreover, several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients (⩾30 mm on VAS) compared with those with moderate to severe pain. This exploratory study provides a molecular basis for the clinical diagnosis of pulpitis. With an enhanced understanding of pulpal inflammation, future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology.

  11. Children's Representations of Facial Expression and Identity: Identity-Contingent Expression Aftereffects

    Science.gov (United States)

    Vida, Mark D.; Mondloch, Catherine J.

    2009-01-01

    This investigation used adaptation aftereffects to examine developmental changes in the perception of facial expressions. Previous studies have shown that adults' perceptions of ambiguous facial expressions are biased following adaptation to intense expressions. These expression aftereffects are strong when the adapting and probe expressions share…

  12. Nonverbal and verbal emotional expression and health.

    Science.gov (United States)

    Berry, D S; Pennebaker, J W

    1993-01-01

    The spontaneous nonverbal expression of emotion is related to immediate reductions in autonomic nervous system activity. Similar changes in specific autonomic channels occur when individuals are encouraged to verbally express their emotions. Indeed, these physiological changes are most likely to occur among individuals who are either verbally or nonverbally highly expressive. These data suggest that when individuals must actively inhibit emotional expression, they are at increased risk for a variety of health problems. Several experiments are summarized which indicate that verbally expressing traumatic experiences by writing or talking improves physical health, enhances immune function, and is associated with fewer medical visits. Although less research is available regarding nonverbal expression, it is also likely that the nonverbal expression of emotion bears some relation to health status. We propose that the effectiveness of many common expressive therapies (e.g., art, music, cathartic) would be enhanced if clients are encouraged to both express their feelings nonverbally and to put their experiences into words.

  13. Misrecognition of facial expressions in delinquents

    Directory of Open Access Journals (Sweden)

    Matsuura Naomi

    2009-09-01

    Full Text Available Abstract Background Previous reports have suggested impairment in facial expression recognition in delinquents, but controversy remains with respect to how such recognition is impaired. To address this issue, we investigated facial expression recognition in delinquents in detail. Methods We tested 24 male adolescent/young adult delinquents incarcerated in correctional facilities. We compared their performances with those of 24 age- and gender-matched control participants. Using standard photographs of facial expressions illustrating six basic emotions, participants matched each emotional facial expression with an appropriate verbal label. Results Delinquents were less accurate in the recognition of facial expressions that conveyed disgust than were control participants. The delinquents misrecognized the facial expressions of disgust as anger more frequently than did controls. Conclusion These results suggest that one of the underpinnings of delinquency might be impaired recognition of emotional facial expressions, with a specific bias toward interpreting disgusted expressions as hostile angry expressions.

  14. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  15. Summer Oral Expression English course

    CERN Multimedia

    2012-01-01

    An English Oral Expression course will take place this summer from 20 August to 29 September.   Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enroll through this link. Please be sure to indicate your planned absences in the comments field so we can schedule the course. If you need more information please send a message to English.training@cern.ch

  16. Summer Oral Expression English course

    CERN Document Server

    2012-01-01

    An English Oral Expression course will take place this summer at some time between 25 June and 28 September. The exact dates will be decided according to the preferences of the students.   Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enroll through this link. Please be sure to indicate your planned absences in the comments field so we can schedule the course. If you need more information please send a message to English.training@cern.ch

  17. Summer Oral Expression English course

    CERN Document Server

    2013-01-01

    An English Oral Expression course will take place this summer at some time between August 19 and October 4.   Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enroll through this link. Please be sure to indicate your planned absences in the comments field so we can schedule the course. If you need more information please send a message to English.training@cern.ch.

  18. Summer Oral Expression English Course

    CERN Multimedia

    HR Department

    2011-01-01

    An English Oral Expression course will take place between 15 August and 30 September 2011. Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enrol through the following link https://cta.cern.ch/cta2/f?p=110:9:1576796470009589::::X_STATUS,XS_COURSE_NAME,XS_PROGRAMME,XS_SUBCATEGORY,X_COURSE_ID,XS_LANGUAGE,XS_SESSION:D,,1,,4368,B, Or contact: Kerstin FUHRMEISTER (70896) Tessa OSBORNE (72957)  

  19. Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko

    2015-12-23

    Background Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue specific expression differences. However, different types of gene expression alteration should have different effects on an organism, the evolutionary forces that act on them might be different, and different types of genes might show different types of differential expression between species. To confirm this, we studied differentially expressed (DE) genes among closely related groups that have extensive gene expression atlases, and clarified characteristics of different types of DE genes including the identification of regulating loci for differential expression using expression quantitative loci (eQTL) analysis data. Results We detected differentially expressed (DE) genes between rice subspecies in five homologous tissues that were verified using japonica and indica transcriptome atlases in public databases. Using the transcriptome atlases, we classified DE genes into two types, global DE genes and changed-tissues DE genes. Global type DE genes were not expressed in any tissues in the atlas of one subspecies, however changed-tissues type DE genes were expressed in both subspecies with different tissue specificity. For the five tissues in the two japonica-indica combinations, 4.6 ± 0.8 and 5.9 ± 1.5 % of highly expressed genes were global and changed-tissues DE genes, respectively. Changed-tissues DE genes varied in number between tissues, increasing linearly with the abundance of tissue specifically expressed genes in the tissue. Molecular evolution of global DE genes was rapid, unlike that of changed-tissues DE genes. Based on gene ontology, global and changed-tissues DE genes were different, having no common GO terms. Expression differences of most global DE genes were regulated by cis-eQTLs. Expression

  20. Helping children express grief through symbolic communication.

    Science.gov (United States)

    Segal, R M

    1984-12-01

    Communication barriers erected by grieving children delay problem resolution. Use of the expressive arts--music, art, and body movement--in symbolic communication helps them to express overwhelming feelings and cope with trauma and stress.

  1. The College Student's Freedom of Expression

    Science.gov (United States)

    Gibbs, Annette

    1974-01-01

    Discussion of means to ensure freedom of expression by college students. Areas of expression noted are student newspapers, lectures by off-campus speakers, freedom to assemble peaceably and freedom to associate. (EK)

  2. Moxibustion upregulates hippocampal progranulin expression

    Directory of Open Access Journals (Sweden)

    Tao Yi

    2016-01-01

    Full Text Available In China, moxibustion is reported to be useful and has few side effects for chronic fatigue syndrome, but its mechanisms are largely unknown. More recently, the focus has been on the wealth of information supporting stress as a factor in chronic fatigue syndrome, and largely concerns dysregulation in the stress-related hypothalamic-pituitary-adrenal axis. In the present study, we aimed to determine the effect of moxibustion on behavioral symptoms in chronic fatigue syndrome rats and examine possible mechanisms. Rats were subjected to a combination of chronic restraint stress and forced swimming to induce chronic fatigue syndrome. The acupoints Guanyuan (CV4 and Zusanli (ST36, bilateral were simultaneously administered moxibustion. Untreated chronic fatigue syndrome rats and normal rats were used as controls. Results from the forced swimming test, open field test, tail suspension test, real-time PCR, enzyme-linked immunosorbent assay, and western blot assay showed that moxibustion treatment decreased mRNA expression of corticotropin-releasing hormone in the hypothalamus, and adrenocorticotropic hormone and corticosterone levels in plasma, and markedly increased progranulin mRNA and protein expression in the hippocampus. These findings suggest that moxibustion may relieve the behavioral symptoms of chronic fatigue syndrome, at least in part, by modulating the hypothalamic-pituitary-adrenal axis and upregulating hippocampal progranulin.

  3. Hepcidin expression in psoriasis patients

    Directory of Open Access Journals (Sweden)

    Nursel Dilek

    2014-01-01

    Full Text Available Background: Iron is an essential nutrient for mammals. Accelerated loss of nutrients through hyperproliferation and desquamation from the skin in psoriasis is known. Hepcidin is an important and recently discovered regulator of iron homeostasis. Aims and Objectives: The present study was undertaken to investigate the hepcidin expression in psoriasis patients. Materials and Methods: We examined peripheral blood cell counts, serum Fe, ferritin, interleukin-6 (IL-6 and hepcidin levels using respectively automated hematology analyzer, Iron assay on the AEROSET system, chemiluminescent microparticle immunoassay with automated analyzer, and enzyme-linked immunosorbent assay. Results: The independent comparison of Fe, ferritin, IL-6 and hepcidin levels in psoriasis patients and control group (healthy volunteers revealed lower Fe and higher IL-6, hepcidin levels in psoriasis patients. No significant difference was seen in the ferritin level between the psoriasis and the control group. Conclusions: We think that studies on hepcidin expression in psoriatic plaques will contribute to our understanding the role of iron and hepcidin in the pathogenesis of psoriasis.

  4. Edix: A Software for Editing Algebraic Expressions.

    OpenAIRE

    Bouhineau , Denis; Nicaud , Jean-François; Pavard , X.

    2001-01-01

    International audience; The paper presents a computer software, called Edix, devoted to the edition of algebraic expressions in their usual 2D representation. At present, many systems display fine algebraic expressions, but the edition of such expressions is weak. Systems like Word and FrameMaker place sub-expressions in too many boxes so that many editing actions are not simple, while usual CAS (computer algebra systems) just use a 1D representation for the edition. Furthermore, Edix allows ...

  5. Kivy and Langer on expressiveness in music

    Directory of Open Access Journals (Sweden)

    van der Schoot Albert

    2013-01-01

    Full Text Available From 1980 onwards, Peter Kivy has put forward that music does not so much express emotions but rather is expressive of emotions. The character of the music does not represent the character or mood of the composer, but reflects his knowledge of emotional life. Unfortunately, Kivy fails to give credit to Susanne Langer, who brought these views to the fore as early as 1942, claiming that the vitality of music lies in expressiveness, not in expression.

  6. The evolution of gene expression in primates

    OpenAIRE

    Tashakkori Ghanbarian, Avazeh

    2015-01-01

    The evolution of a gene’s expression profile is commonly assumed to be independent of its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between expression of neighboring genes in extant taxa. Indeed, in all eukaryotic genomes, genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their e...

  7. Intrahepatic expression of interferon alpha & interferon alpha ...

    African Journals Online (AJOL)

    kemrilib

    IFN-α and IFN-α Receptor mRNA expression in the liver. All the patients showed IFN-α gene expression except one patient who had the highest degree of fibrosis (fibrosis grade 5) and HAI Index of 9. IFN- α Receptor mRNA was expressed in 30% (9/30). (Figure 4). Non of the patients with HCC had. IFNα-Rc expression and ...

  8. Generating Expressive Speech for Storytelling Applications

    NARCIS (Netherlands)

    Bailly, G.; Theune, Mariet; Meijs, Koen; Campbell, N.; Hamza, W.; Heylen, Dirk K.J.; Ordelman, Roeland J.F.; Hoge, H.; Jianhua, T.

    Work on expressive speech synthesis has long focused on the expression of basic emotions. In recent years, however, interest in other expressive styles has been increasing. The research presented in this paper aims at the generation of a storytelling speaking style, which is suitable for

  9. 75 FR 473 - Community Express Pilot Program

    Science.gov (United States)

    2010-01-05

    ... SMALL BUSINESS ADMINISTRATION Community Express Pilot Program AGENCY: U.S. Small Business Administration (SBA). ACTION: Notice of extension of the Community Express Pilot Program. SUMMARY: This notice extends the Community Express Pilot Program in its current form through December 31, 2010. Based upon the...

  10. 75 FR 80561 - Community Express Pilot Program

    Science.gov (United States)

    2010-12-22

    ... SMALL BUSINESS ADMINISTRATION Community Express Pilot Program AGENCY: U.S. Small Business Administration (SBA). ACTION: Notice of short-term extension and termination of the Community Express Pilot Program. SUMMARY: This notice announces the termination of the Community Express Pilot Program following a...

  11. A constructive approach to gene expression dynamics

    International Nuclear Information System (INIS)

    Ochiai, T.; Nacher, J.C.; Akutsu, T.

    2004-01-01

    Recently, experiments on mRNA abundance (gene expression) have revealed that gene expression shows a stationary organization described by a scale-free distribution. Here we propose a constructive approach to gene expression dynamics which restores the scale-free exponent and describes the intermediate state dynamics. This approach requires only one assumption: Markov property

  12. Vocal Emotion Expressions Effects on Cooperation Behavior

    Science.gov (United States)

    Caballero Meneses, Jonathan Azael; Menez Díaz, Judith Marina

    2017-01-01

    Emotional expressions have been proposed to be important for regulating social interaction as they can serve as cues for behavioral intentions. The issue has been mainly addressed analyzing the effects of facial emotional expressions in cooperation behavior, but there are contradictory results regarding the impact of emotional expressions on that…

  13. Construction of expression vectors carrying mouse peroxisomal ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-16

    Nov 16, 2009 ... The aim of this study was to construct expression vectors carrying mouse peroxisomal protein gene. (PEP-cDNA) in prokaryotic and mammalian expression vectors in chimeric cDNA types, encompassing. GST and FLAG with PEP-cDNA. PEP-cDNA was sub-cloned in pGEX6p2 prokaryotic expression ...

  14. Endogenous retrovirus sequences expressed in male mammalian ...

    African Journals Online (AJOL)

    In humans, one ERV family, human endogenous retrovirus- K (HERV-K) is abundantly expressed, and is associated with germ cell tumours, while ERV3 env is expressed in normal human testis. Conclusion: The expression of ERVs in male reproductive tissues suggests a possible role in normal and disease conditions ...

  15. Expressiveness modulo Bisimilarity of Regular Expressions with Parallel Composition (Extended Abstract

    Directory of Open Access Journals (Sweden)

    Jos C. M. Baeten

    2010-11-01

    Full Text Available The languages accepted by finite automata are precisely the languages denoted by regular expressions. In contrast, finite automata may exhibit behaviours that cannot be described by regular expressions up to bisimilarity. In this paper, we consider extensions of the theory of regular expressions with various forms of parallel composition and study the effect on expressiveness. First we prove that adding pure interleaving to the theory of regular expressions strictly increases its expressiveness up to bisimilarity. Then, we prove that replacing the operation for pure interleaving by ACP-style parallel composition gives a further increase in expressiveness. Finally, we prove that the theory of regular expressions with ACP-style parallel composition and encapsulation is expressive enough to express all finite automata up to bisimilarity. Our results extend the expressiveness results obtained by Bergstra, Bethke and Ponse for process algebras with (the binary variant of Kleene's star operation.

  16. Recognising and Interpreting Named Temporal Expressions

    DEFF Research Database (Denmark)

    Brucato, Matteo; Derczynski, Leon; Llorens, Hectjor

    2013-01-01

    This paper introduces a new class of temporal expression – named temporal expressions – and methods for recognising and interpreting its members. The commonest temporal expressions typically contain date and time words, like April or hours. Research into recognising and interpreting these typical...... expressions is mature in many languages. However, there is a class of expressions that are less typical, very varied, and difficult to automatically interpret. These indicate dates and times, but are harder to detect because they often do not contain time words and are not used frequently enough to appear...

  17. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    knockout and strong overexpression. However, applications such as metabolic optimization and control analysis necessitate a continuous set of expression levels with only slight increments in strength to cover a specific window around the wildtype expression level of the studied gene; this requirement can......The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene...

  18. Expressing Model Constraints Visually with VMQL

    DEFF Research Database (Denmark)

    Störrle, Harald

    2011-01-01

    ) for specifying constraints on UML models. We examine VMQL's usability by controlled experiments and its expressiveness by a representative sample. We conclude that VMQL is less expressive than OCL, although expressive enough for most of the constraints in the sample. In terms of usability, however, VMQL......OCL is the de facto standard language for expressing constraints and queries on UML models. However, OCL expressions are very difficult to create, understand, and maintain, even with the sophisticated tool support now available. In this paper, we propose to use the Visual Model Query Language (VMQL...

  19. Abstract Expression Grammar Symbolic Regression

    Science.gov (United States)

    Korns, Michael F.

    This chapter examines the use of Abstract Expression Grammars to perform the entire Symbolic Regression process without the use of Genetic Programming per se. The techniques explored produce a symbolic regression engine which has absolutely no bloat, which allows total user control of the search space and output formulas, which is faster, and more accurate than the engines produced in our previous papers using Genetic Programming. The genome is an all vector structure with four chromosomes plus additional epigenetic and constraint vectors, allowing total user control of the search space and the final output formulas. A combination of specialized compiler techniques, genetic algorithms, particle swarm, aged layered populations, plus discrete and continuous differential evolution are used to produce an improved symbolic regression sytem. Nine base test cases, from the literature, are used to test the improvement in speed and accuracy. The improved results indicate that these techniques move us a big step closer toward future industrial strength symbolic regression systems.

  20. E-mail: Outlook Express

    Directory of Open Access Journals (Sweden)

    Zainul Bakri

    2012-10-01

    Full Text Available Salah satu layanan Internet yang sangat penting adalah electronic mail atau sering hanya disebut sebagai e-mail. Untuk menggunakan e-mail, diperlukan piranti lunak khusus supaya pengguna dapat mengirim dan menerima e-mail. Jenis piranti lunak e-mail diantaranya adalah Outlook Express yang merupakan satu paket yang didistribusikan bersama Internet Explorer versi 4. Piranti lunak ini dijalankan pada PC yang mempunyai sistem operasi Windows 95 atau 98. Jenis piranti lunak e-mail yang lain adalah Eudora, Pegasus dan sebagainya. Bahkan ada yang diintegrasikan dengan Web Browser (alat untuk menelusuri situs Web misalnya IE,dan Netscape.Sebagai layaknya pelayanan pos, maka setiap pengguna e-mail mempunyai alamat tertentu yang tidak mungkin dipunyai oleh pengguna lainnya diseluruh dunia. Untuk keperluan pendistribusian, maka e-mail mempunyai semacam kantor pos yang ditempatkan dalam sebuah komputer server (mail server atau sering disebut sebagai host. 

  1. Generalized Expression for Polarization Density

    International Nuclear Information System (INIS)

    Wang, Lu; Hahm, T.S.

    2009-01-01

    A general polarization density which consists of classical and neoclassical parts is systematically derived via modern gyrokinetics and bounce-kinetics by employing a phase-space Lagrangian Lie-transform perturbation method. The origins of polarization density are further elucidated. Extending the work on neoclassical polarization for long wavelength compared to ion banana width [M. N. Rosenbluth and F. L. Hinton, Phys. Rev. Lett. 80, 724 (1998)], an analytical formula for the generalized neoclassical polarization including both finite-banana-width (FBW) and finite-Larmor-radius (FLR) effects for arbitrary radial wavelength in comparison to banana width and gyroradius is derived. In additional to the contribution from trapped particles, the contribution of passing particles to the neoclassical polarization is also explicitly calculated. Our analytic expression agrees very well with the previous numerical results for a wide range of radial wavelength.

  2. Perceived intimacy of expressed emotion.

    Science.gov (United States)

    Howell, A; Conway, M

    1990-08-01

    Research on norms for emotional expression and self-disclosure provided the basis for two hypotheses concerning the perceived intimacy of emotional self-disclosure. The first hypothesis was that the perceived intimacy of negative emotional disclosure would be greater than that of positive emotional disclosure; the second was that disclosures of more intense emotional states would be perceived as more intimate than disclosures of less intense emotional states for both negative and positive disclosures. Both hypotheses received support when male students in Canada rated the perceived intimacy of self-disclosures that were equated for topic and that covered a comprehensive sample of emotions and a range of emotional intensities. The effects were observed across all the topics of disclosure examined.

  3. Podoplanin Expression in Canine Melanoma.

    Science.gov (United States)

    Ogasawara, Satoshi; Honma, Ryusuke; Kaneko, Mika K; Fujii, Yuki; Kagawa, Yumiko; Konnai, Satoru; Kato, Yukinari

    2016-12-01

    A type I transmembrane protein, podoplanin (PDPN), is expressed in several normal cells such as lymphatic endothelial cells or pulmonary type I alveolar cells. We recently demonstrated that anticanine PDPN monoclonal antibody (mAb), PMab-38, recognizes canine PDPN of squamous cell carcinomas, but does not react with lymphatic endothelial cells. Herein, we investigated whether PMab-38 reacts with canine melanoma. PMab-38 reacted with 90% of melanoma cells (9/10 cases) using immunohistochemistry. Of interest, PMab-38 stained the lymphatic endothelial cells and cancer-associated fibroblasts in melanoma tissues, although it did not stain any lymphatic endothelial cells in normal tissues. PMab-38 could be useful for uncovering the function of PDPN in canine melanomas.

  4. Definition, Detection and Generation of Iyashi Expressions

    Science.gov (United States)

    Kitaoka, Tetsuko; Diago, Luis A.; Hagiwara, Ichiro; Kitazaki, Satoshi; Yamane, Shigeru

    This paper concerns the engineering analysis of “Iyashi”, a peculiar concept to the Japanese, which affect person's heart and may change their expression and behavior. We have integrated the advocator's view of “Iyashi”, analyzed the social background of “Iyashi” and have defined Iyashi and also the Iyashi expression. As the facial expression is the special and important stimulus for both observers and people who show expressions, we want to prove the existence of expressions that change the observer's emotion with Iyashi. We have developed the system to clarify the combination of facial features important for Iyashi through the psychological experiments and the analysis by Holographic Neural Networks (HNN). HNN analysis gave the structure of the Iyashi expression, that is the important combination of the physical facial parameters contributing to the high degree of Iyashi. Based on the structure of Iyashi we are able to generate the Iyashi expression appropriate for each person.

  5. Adaptive Evolution of Gene Expression in Drosophila

    Directory of Open Access Journals (Sweden)

    Armita Nourmohammad

    2017-08-01

    Full Text Available Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. Here, we present evidence that adaptation dominates the evolution of gene expression levels in flies. We show that 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. Our results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, we identify a large group of genes with sex-specific adaptation of expression, which predominantly occurs in males. Our analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis.

  6. cGMP and NHR signaling co-regulate expression of insulin-like peptides and developmental activation of infective larvae in Strongyloides stercoralis.

    Directory of Open Access Journals (Sweden)

    Jonathan D Stoltzfus

    2014-07-01

    Full Text Available The infectious form of the parasitic nematode Strongyloides stercoralis is a developmentally arrested third-stage larva (L3i, which is morphologically similar to the developmentally arrested dauer larva in the free-living nematode Caenorhabditis elegans. We hypothesize that the molecular pathways regulating C. elegans dauer development also control L3i arrest and activation in S. stercoralis. This study aimed to determine the factors that regulate L3i activation, with a focus on G protein-coupled receptor-mediated regulation of cyclic guanosine monophosphate (cGMP pathway signaling, including its modulation of the insulin/IGF-1-like signaling (IIS pathway. We found that application of the membrane-permeable cGMP analog 8-bromo-cGMP potently activated development of S. stercoralis L3i, as measured by resumption of feeding, with 85.1 ± 2.2% of L3i feeding in 200 µM 8-bromo-cGMP in comparison to 0.6 ± 0.3% in the buffer diluent. Utilizing RNAseq, we examined L3i stimulated with DMEM, 8-bromo-cGMP, or the DAF-12 nuclear hormone receptor (NHR ligand Δ7-dafachronic acid (DA--a signaling pathway downstream of IIS in C. elegans. L3i stimulated with 8-bromo-cGMP up-regulated transcripts of the putative agonistic insulin-like peptide (ILP -encoding genes Ss-ilp-1 (20-fold and Ss-ilp-6 (11-fold in comparison to controls without stimulation. Surprisingly, we found that Δ7-DA similarly modulated transcript levels of ILP-encoding genes. Using the phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitor LY294002, we demonstrated that 400 nM Δ7-DA-mediated activation (93.3 ± 1.1% L3i feeding can be blocked using this IIS inhibitor at 100 µM (7.6 ± 1.6% L3i feeding. To determine the tissues where promoters of ILP-encoding genes are active, we expressed promoter::egfp reporter constructs in transgenic S. stercoralis post-free-living larvae. Ss-ilp-1 and Ss-ilp-6 promoters are active in the hypodermis and neurons and the Ss-ilp-7 promoter is active in the

  7. Easy measurement of diffusion coefficients of EGFP-tagged plasma membrane proteins using k-space Image Correlation Spectroscopy

    DEFF Research Database (Denmark)

    Christensen, Eva Arnspang; Koffman, Jennifer Skaarup; Marlar, Saw

    2014-01-01

    Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1 was developed to enable...... routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely...... to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS. First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope Then...

  8. Polyomavirus EGFP-pseudocapsids:analysis of model particles for introduction of proteins and pepetides into mammalian cells

    Czech Academy of Sciences Publication Activity Database

    Bouřa, E.; Liebl, D.; Spíšek, R.; Frič, Jan; Marek, M.; Štokrová, Jitka; Holáň, Vladimír; Forstová, J.

    2005-01-01

    Roč. 579, č. 29 (2005), s. 6549-6558 ISSN 0014-5793 R&D Projects: GA MZd(CZ) NC6957; GA ČR(CZ) GA204/03/0593 Institutional research plan: CEZ:AV0Z50520514 Keywords : mouse polyomavirus * empty artificial virus-like particle * dendritic cell activation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.415, year: 2005

  9. Anticausatives are weak scalar expressions, not reflexive expressions

    Directory of Open Access Journals (Sweden)

    Florian Schäfer

    2016-07-01

    Full Text Available We discuss conceptual and empirical arguments from Germanic, Romance and Slavic languages against an analysis treating anticausative verbs as derived from their lexical causative counterparts under reflexivization. Instead, we defend the standard account to the semantics of the causative alternation according to which anticausatives in general, and anticausatives marked with reflexive morphology in particular, denote simple one-place inchoative events that are logically entailed by their lexical causative counterparts. Under such an account, anticausative verbs are weak scalar expressions that stand in a semantico-pragmatic opposition to their strong lexical causative counterparts. Due to this scalar relation, the use of an anticausative can trigger the implicature that the use of its lexical causative counterpart is too strong. As usual with implicatures, they can be ‘metalinguistically’ denied, cancelled, or reinforced and we argue that these mechanisms explain all central empirical facts brought up in the literature in favor of a treatment of anticausatives as semantically reflexive predicates. Our results reinforce the view that the reflexive morphemes used in many (Indo-European languages to mark anticausatives do not necessarily trigger reflexive semantics. However, we also show that a string involving a reflexively marked (anti-causative verb can be forced into a semantically reflexive construal under particular conceptual or grammatical circumstances.

  10. Cyclooxygenase-2 expression in the normal human eye and its expression pattern in selected eye tumours

    DEFF Research Database (Denmark)

    Wang, Jinmei; Wu, Yazhen; Heegaard, Steffen

    2011-01-01

    Purpose: Cyclooxygenase-2 (COX-2) is an enzyme involved in neoplastic processes. The purpose of the present study is to investigate COX-2 expression in the normal human eye and the expression pattern in selected eye tumours involving COX-2 expressing cells. Methods: Immunohistochemical staining...... using antibodies against COX-2 was performed on paraffin sections of normal human eyes and selected eye tumours arising from cells expressing COX-2. Results: Cyclooxygenase-2 expression was found in various structures of the normal eye. Abundant expression was seen in the cornea, iris, ciliary body...... and retina. The COX-2 expression was less in tumours deriving from the ciliary epithelium and also in retinoblastoma. Conclusion: Cyclooxygenase-2 is constitutively expressed in normal human eyes. The expression of COX-2 is much lower in selected eye tumours involving COX-2 expressing cells....

  11. A study of the dynamics of PTEN proteins in living cells using in vivo fluorescence correlation spectroscopy

    Science.gov (United States)

    Du, Zhixue; Dong, Chaoqing; Ren, Jicun

    2017-06-01

    PTEN (phosphatase and tensin homolog on chromosome 10) is one of the most important tumor-suppressor proteins, which plays a key role in negative regulation of the PI3K/AKT pathway, and governs many cellular processes including growth, proliferation, survival and migration. The dynamics of PTEN proteins in single living cells is as yet unclear owing to a shortage of suitable in vivo approaches. Here, we report a single-molecule method for in vivo study of the dynamics of PTEN proteins in living cells using fluorescence correlation spectroscopy (FCS). First, we established a monoclonal H1299 stable cell line expressing enhanced green fluorescent protein (EGFP) and PTEN (EGFP-PTEN) fusion proteins; we then developed an in vivo FCS method to study the dynamics of EGFP-PTEN both in the nucleus and the cytoplasm. We investigated the diffusion behaviors of EGFP and EGFP-PTEN in solution, nucleus and cytosol, and observed that the motion of PTEN in living cells was restricted compared with EGFP. Finally, we investigated the protein dynamics in living cells under oxidative stress stimulation and a cellular ATP depletion treatment. Under oxidative stress stimulation, the EGFP-PTEN concentration increased in the nucleus, but slightly decreased in the cytoplasm. The diffusion coefficient and alpha value of EGFP-PTEN reduced significantly both in the nucleus and cytoplasm; the significantly decreased alpha parameter indicates a more restricted Brownian diffusion behavior. Under the cellular ATP depletion treatment, the concentration of EGFP-PTEN remained unchanged in the nucleus and decreased significantly in cytosol. The diffusion coefficient of EGFP-PTEN decreased significantly in cytosol, but showed no significant change in the nucleus; the alpha value decreased significantly in both the nucleus and cytoplasm. These results suggest that the concentration and mobility of PTEN in the nucleus and cytoplasm can be regulated by stimulation methods. Our approach provides a unique

  12. Facial expressions and pair bonds in hylobatids.

    Science.gov (United States)

    Florkiewicz, Brittany; Skollar, Gabriella; Reichard, Ulrich H

    2018-06-06

    Facial expressions are an important component of primate communication that functions to transmit social information and modulate intentions and motivations. Chimpanzees and macaques, for example, produce a variety of facial expressions when communicating with conspecifics. Hylobatids also produce various facial expressions; however, the origin and function of these facial expressions are still largely unclear. It has been suggested that larger facial expression repertoires may have evolved in the context of social complexity, but this link has yet to be tested at a broader empirical basis. The social complexity hypothesis offers a possible explanation for the evolution of complex communicative signals such as facial expressions, because as the complexity of an individual's social environment increases so does the need for communicative signals. We used an intraspecies, pair-focused study design to test the link between facial expressions and sociality within hylobatids, specifically the strength of pair-bonds. The current study compared 206 hr of video and 103 hr of focal animal data for ten hylobatid pairs from three genera (Nomascus, Hoolock, and Hylobates) living at the Gibbon Conservation Center. Using video footage, we explored 5,969 facial expressions along three dimensions: repertoire use, repertoire breadth, and facial expression synchrony [FES]. We then used focal animal data to compare dimensions of facial expressiveness to pair bond strength and behavioral synchrony. Hylobatids in our study overlapped in only half of their facial expressions (50%) with the only other detailed, quantitative study of hylobatid facial expressions, while 27 facial expressions were uniquely observed in our study animals. Taken together, hylobatids have a large facial expression repertoire of at least 80 unique facial expressions. Contrary to our prediction, facial repertoire composition was not significantly correlated with pair bond strength, rates of territorial synchrony

  13. Expressiveness in musical performance: Pedagogic aspect

    Directory of Open Access Journals (Sweden)

    Jović Natalija R.

    2016-01-01

    Full Text Available The subject of our research relates to pedagogic aspects of expressive vocal-instrumental musical performance. We intended to examine: (1 how undergraduate students see/conceptualize and evaluate expressiveness in musical performance; (2 whether and how they were trained in the skill of expressive musical performance during their musical training; (3 whether and in which way they rehearse the expressive component of musical performance and interpretation and (4 whether there are any differences regarding gender, age, instrument, department, year of study and years of instrument playing in relation to the group of dependant variables related to expressiveness, tuition and practice. The sample for the research included 82 students of instrumental and theory departments at the Faculty of Music in Belgrade. Psychological and pedagogical aspects of musical expressiveness during vocal-instrumental performance were analyzed. The results show that students highly evaluate expressiveness but its place is secondary compared to mastering technical and tonal requirements. Statistically significant differences were shown regarding gender, age and departments. It can be concluded that there is a potential for the development and enhancement of expressiveness of students if we abandon the traditional view that expressiveness is linked exclusively to talent. The findings indicate that pedagogical work should be directed towards finding purposeful strategies for training individual expressiveness.

  14. Widespread ectopic expression of olfactory receptor genes

    Directory of Open Access Journals (Sweden)

    Yanai Itai

    2006-05-01

    Full Text Available Abstract Background Olfactory receptors (ORs are the largest gene family in the human genome. Although they are expected to be expressed specifically in olfactory tissues, some ectopic expression has been reported, with special emphasis on sperm and testis. The present study systematically explores the expression patterns of OR genes in a large number of tissues and assesses the potential functional implication of such ectopic expression. Results We analyzed the expression of hundreds of human and mouse OR transcripts, via EST and microarray data, in several dozens of human and mouse tissues. Different tissues had specific, relatively small OR gene subsets which had particularly high expression levels. In testis, average expression was not particularly high, and very few highly expressed genes were found, none corresponding to ORs previously implicated in sperm chemotaxis. Higher expression levels were more common for genes with a non-OR genomic neighbor. Importantly, no correlation in expression levels was detected for human-mouse orthologous pairs. Also, no significant difference in expression levels was seen between intact and pseudogenized ORs, except for the pseudogenes of subfamily 7E which has undergone a human-specific expansion. Conclusion The OR superfamily as a whole, show widespread, locus-dependent and heterogeneous expression, in agreement with a neutral or near neutral evolutionary model for transcription control. These results cannot reject the possibility that small OR subsets might play functional roles in different tissues, however considerable care should be exerted when offering a functional interpretation for ectopic OR expression based only on transcription information.

  15. Platelet cyclooxygenase expression in normal dogs.

    Science.gov (United States)

    Thomason, J; Lunsford, K; Mullins, K; Stokes, J; Pinchuk, L; Wills, R; McLaughlin, R; Langston, C; Pruett, S; Mackin, A

    2011-01-01

    Human platelets express both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Variation in COX-2 expression could be a mechanism for variable response to aspirin. The hypotheses were that circulating canine platelets express COX-1 and COX-2, and that aspirin alters COX expression. The objective was to identify changes in platelet COX expression and in platelet function caused by aspirin administration to dogs. Eight female, intact hounds. A single population, repeated measures design was used to evaluate platelet COX-1 and COX-2 expression by flow cytometry before and after aspirin (10 mg/kg Q12h for 10 days). Platelet function was analyzed via PFA-100(®) (collagen/epinephrine), and urine 11-dehydro-thromboxane B(2) (11-dTXB(2)) was measured and normalized to urinary creatinine. Differences in COX expression, PFA-100(®) closure times, and urine 11-dTXB(2 ): creatinine ratio were analyzed before and after aspirin administration. Both COX-1 and COX-2 were expressed in canine platelets. COX-1 mean fluorescent intensity (MFI) increased in all dogs, by 250% (range 63-476%), while COX-2 expression did not change significantly (P = 0.124) after aspirin exposure, with large interindividual variation. PFA-100(®) closure times were prolonged and urine 11-dTXB(2) concentration decreased in all dogs after aspirin administration. Canine platelets express both COX isoforms. After aspirin exposure, COX-1 expression increased despite impairment of platelet function, while COX-2 expression varied markedly among dogs. Variability in platelet COX-2 expression should be explored as a potential mechanism for, or marker of, variable aspirin responsiveness. Copyright © 2011 by the American College of Veterinary Internal Medicine.

  16. Altered balance of glutamatergic/GABAergic synaptic input and associated changes in dendrite morphology after BDNF expression in BDNF-deficient hippocampal neurons

    OpenAIRE

    Singh, B.; Henneberger, C.; Betances, D.; Arevalo, M.A.; Rodriguez-Tebar, A.; Meier, J.C.; Grantyn, R.

    2006-01-01

    Cultured neurons from bdnf-/- mice display reduced densities of synaptic terminals, although in vivo these deficits are small or absent. Here we aimed at clarifying the local responses to postsynaptic brain-derived neurotrophic factor (BDNF). To this end, solitary enhanced green fluorescent protein (EGFP)-labeled hippocampal neurons from bdnf-/- mice were compared with bdnf-/- neurons after transfection with BDNF, bdnf-/- neurons after transient exposure to exogenous BDNF, and bdnf+/+ neurons...

  17. Expressiveness and definability in circumscription

    Directory of Open Access Journals (Sweden)

    Francicleber Martins Ferreira

    2011-06-01

    Full Text Available We investigate expressiveness and definability issues with respect to minimal models, particularly in the scope of Circumscription. First, we give a proof of the failure of the Löwenheim-Skolem Theorem for Circumscription. Then we show that, if the class of P; Z-minimal models of a first-order sentence is Δ-elementary, then it is elementary. That is, whenever the circumscription of a first-order sentence is equivalent to a first-order theory, then it is equivalent to a finitely axiomatizable one. This means that classes of models of circumscribed theories are either elementary or not Δ-elementary. Finally, using the previous result, we prove that, whenever a relation Pi is defined in the class of P; Z-minimal models of a first-order sentence Φ and whenever such class of P; Z-minimal models is Δ-elementary, then there is an explicit definition ψ for Pi such that the class of P; Z-minimal models of Φ is the class of models of Φ ∧ ψ. In order words, the circumscription of P in Φ with Z varied can be replaced by Φ plus this explicit definition ψ for Pi.

  18. Adipocyte differentiation and leptin expression

    DEFF Research Database (Denmark)

    Hwang, C S; Loftus, T M; Mandrup, S

    1997-01-01

    Adipose tissue has long been known to house the largest energy reserves in the animal body. Recent research indicates that in addition to this role, the adipocyte functions as a global regulator of energy metabolism. Adipose tissue is exquisitely sensitive to a variety of endocrine and paracrine ...... of energy intake and expenditure. The hormonal and transcriptional control of adipocyte differentiation is discussed, as is the role of leptin and other factors secreted by the adipocyte that participate in the regulation of adipose homeostasis.......Adipose tissue has long been known to house the largest energy reserves in the animal body. Recent research indicates that in addition to this role, the adipocyte functions as a global regulator of energy metabolism. Adipose tissue is exquisitely sensitive to a variety of endocrine and paracrine......, most notably those of the C/EBP and PPAR families, which combine to regulate each other and to control the expression of adipocyte-specific genes. One such gene, i.e. the obese gene, was recently identified and found to encode a hormone, referred to as leptin, that plays a major role in the regulation...

  19. Recognizing Facial Expressions Automatically from Video

    Science.gov (United States)

    Shan, Caifeng; Braspenning, Ralph

    Facial expressions, resulting from movements of the facial muscles, are the face changes in response to a person's internal emotional states, intentions, or social communications. There is a considerable history associated with the study on facial expressions. Darwin [22] was the first to describe in details the specific facial expressions associated with emotions in animals and humans, who argued that all mammals show emotions reliably in their faces. Since that, facial expression analysis has been a area of great research interest for behavioral scientists [27]. Psychological studies [48, 3] suggest that facial expressions, as the main mode for nonverbal communication, play a vital role in human face-to-face communication. For illustration, we show some examples of facial expressions in Fig. 1.

  20. Stochastic gene expression in Arabidopsis thaliana.

    Science.gov (United States)

    Araújo, Ilka Schultheiß; Pietsch, Jessica Magdalena; Keizer, Emma Mathilde; Greese, Bettina; Balkunde, Rachappa; Fleck, Christian; Hülskamp, Martin

    2017-12-14

    Although plant development is highly reproducible, some stochasticity exists. This developmental stochasticity may be caused by noisy gene expression. Here we analyze the fluctuation of protein expression in Arabidopsis thaliana. Using the photoconvertible KikGR marker, we show that the protein expressions of individual cells fluctuate over time. A dual reporter system was used to study extrinsic and intrinsic noise of marker gene expression. We report that extrinsic noise is higher than intrinsic noise and that extrinsic noise in stomata is clearly lower in comparison to several other tissues/cell types. Finally, we show that cells are coupled with respect to stochastic protein expression in young leaves, hypocotyls and roots but not in mature leaves. Our data indicate that stochasticity of gene expression can vary between tissues/cell types and that it can be coupled in a non-cell-autonomous manner.

  1. Osteopontin expression in salivary gland carcinomas

    DEFF Research Database (Denmark)

    Bjørndal, Kristine; Larsen, Stine R; Godballe, Christian

    2011-01-01

    J Oral Pathol Med (2010) Background:  In several cancer types, osteopontin (OPN) expression has been correlated with tumor progression and prognosis. Two earlier studies have examined OPN expression in salivary gland carcinomas with contradictory results. Methods:  One hundred and seventy......:  Osteopontin was expressed in all salivary gland carcinomas. Adenoid cystic carcinomas had the highest mean sum score (7.3) and a significantly higher proportion of carcinomas with high OPN sum score than both mucoepidermoid carcinoma and acinic cell carcinoma. Correlation of OPN expression with known...... prognostic factors in salivary gland carcinomas was insignificant. Conclusions:  Salivary gland carcinomas express OPN. The expression does not correlate with known prognostic factors....

  2. Chemical coding and chemosensory properties of cholinergic brush cells in the mouse gastrointestinal and biliary tract.

    Science.gov (United States)

    Schütz, Burkhard; Jurastow, Innokentij; Bader, Sandra; Ringer, Cornelia; von Engelhardt, Jakob; Chubanov, Vladimir; Gudermann, Thomas; Diener, Martin; Kummer, Wolfgang; Krasteva-Christ, Gabriela; Weihe, Eberhard

    2015-01-01

    The mouse gastro-intestinal and biliary tract mucosal epithelia harbor choline acetyltransferase (ChAT)-positive brush cells with taste cell-like traits. With the aid of two transgenic mouse lines that express green fluorescent protein (EGFP) under the control of the ChAT promoter (EGFP (ChAT) ) and by using in situ hybridization and immunohistochemistry we found that EGFP (ChAT) cells were clustered in the epithelium lining the gastric groove. EGFP (ChAT) cells were numerous in the gall bladder and bile duct, and found scattered as solitary cells along the small and large intestine. While all EGFP (ChAT) cells were also ChAT-positive, expression of the high-affinity choline transporter (ChT1) was never detected. Except for the proximal colon, EGFP (ChAT) cells also lacked detectable expression of the vesicular acetylcholine transporter (VAChT). EGFP (ChAT) cells were found to be separate from enteroendocrine cells, however they were all immunoreactive for cytokeratin 18 (CK18), transient receptor potential melastatin-like subtype 5 channel (TRPM5), and for cyclooxygenases 1 (COX1) and 2 (COX2). The ex vivo stimulation of colonic EGFP (ChAT) cells with the bitter substance denatonium resulted in a strong increase in intracellular calcium, while in other epithelial cells such an increase was significantly weaker and also timely delayed. Subsequent stimulation with cycloheximide was ineffective in both cell populations. Given their chemical coding and chemosensory properties, EGFP (ChAT) brush cells thus may have integrative functions and participate in induction of protective reflexes and inflammatory events by utilizing ACh and prostaglandins for paracrine signaling.

  3. "Express yourself": culture and the effect of self-expression on choice.

    Science.gov (United States)

    Kim, Heejung S; Sherman, David K

    2007-01-01

    Whereas self-expression is valued in the United States, it is not privileged with such a cultural emphasis in East Asia. Four studies demonstrate the psychological implications of this cultural difference. Studies 1 and 2 found that European Americans value self-expression more than East Asians/East Asian Americans. Studies 3 and 4 examined the roles of expression in preference judgments. In Study 3, the expression of choice led European Americans but not East Asian Americans to be more invested in what they chose. Study 4 examined the connection between the value of expression and the effect of choice expression and showed that European Americans place greater emphasis on self-expression than East Asian Americans, and this difference explained the cultural difference in Study 3. This research highlights the importance of the cultural meanings of self-expression and the moderating role of cultural beliefs on the psychological effect of self-expression. 2007 APA, all rights reserved

  4. Generating Expressive Speech for Storytelling Applications

    OpenAIRE

    Bailly, G.; Theune, Mariet; Meijs, Koen; Campbell, N.; Hamza, W.; Heylen, Dirk K.J.; Ordelman, Roeland J.F.; Hoge, H.; Jianhua, T.

    2006-01-01

    Work on expressive speech synthesis has long focused on the expression of basic emotions. In recent years, however, interest in other expressive styles has been increasing. The research presented in this paper aims at the generation of a storytelling speaking style, which is suitable for storytelling applications and more in general, for applications aimed at children. Based on an analysis of human storytellers' speech, we designed and implemented a set of prosodic rules for converting "neutr...

  5. Willingness to Express Emotions to Caregiving Spouses

    OpenAIRE

    Monin, Joan K.; Martire, Lynn M.; Schulz, Richard; Clark, Margaret S.

    2009-01-01

    This study examined the association between care-recipients’ willingness to express emotions to spousal caregivers and caregiver’s well-being and support behaviors. Using self-report measures in the context of a larger study, 262 care-recipients with osteoarthritis reported on their willingness to express emotions to caregivers, and caregivers reported on their stress and insensitive responding to care-recipients. Results revealed that care-recipients’ willingness to express happiness was ass...

  6. Comparative gene expression between two yeast species

    Directory of Open Access Journals (Sweden)

    Guan Yuanfang

    2013-01-01

    Full Text Available Abstract Background Comparative genomics brings insight into sequence evolution, but even more may be learned by coupling sequence analyses with experimental tests of gene function and regulation. However, the reliability of such comparisons is often limited by biased sampling of expression conditions and incomplete knowledge of gene functions across species. To address these challenges, we previously systematically generated expression profiles in Saccharomyces bayanus to maximize functional coverage as compared to an existing Saccharomyces cerevisiae data repository. Results In this paper, we take advantage of these two data repositories to compare patterns of ortholog expression in a wide variety of conditions. First, we developed a scalable metric for expression divergence that enabled us to detect a significant correlation between sequence and expression conservation on the global level, which previous smaller-scale expression studies failed to detect. Despite this global conservation trend, between-species gene expression neighborhoods were less well-conserved than within-species comparisons across different environmental perturbations, and approximately 4% of orthologs exhibited a significant change in co-expression partners. Furthermore, our analysis of matched perturbations collected in both species (such as diauxic shift and cell cycle synchrony demonstrated that approximately a quarter of orthologs exhibit condition-specific expression pattern differences. Conclusions Taken together, these analyses provide a global view of gene expression patterns between two species, both in terms of the conditions and timing of a gene's expression as well as co-expression partners. Our results provide testable hypotheses that will direct future experiments to determine how these changes may be specified in the genome.

  7. Structural development of child's artistic expression

    OpenAIRE

    Sanja Filipović; Milica Vojvodić

    2017-01-01

    Abstract Structural development implies control and capability of the expression usage in terms of independent creative expression and making. Understanding of structural development of child's artistic expression as a phenomenon (which is suitable to child's age) has some implications on methodical acts considering artistic education of children and youngsters. Therefore, it is of unexceptional importance to know these laws as well as methodical acts which encourage the structural develop...

  8. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy [Davis, CA; Bachkirova, Elena [Davis, CA; Rey, Michael [Davis, CA

    2012-05-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  9. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2013-10-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  10. Identification of Emotional Facial Expressions: Effects of Expression, Intensity, and Sex on Eye Gaze.

    Directory of Open Access Journals (Sweden)

    Laura Jean Wells

    Full Text Available The identification of emotional expressions is vital for social interaction, and can be affected by various factors, including the expressed emotion, the intensity of the expression, the sex of the face, and the gender of the observer. This study investigates how these factors affect the speed and accuracy of expression recognition, as well as dwell time on the two most significant areas of the face: the eyes and the mouth. Participants were asked to identify expressions from female and male faces displaying six expressions (anger, disgust, fear, happiness, sadness, and surprise, each with three levels of intensity (low, moderate, and normal. Overall, responses were fastest and most accurate for happy expressions, but slowest and least accurate for fearful expressions. More intense expressions were also classified most accurately. Reaction time showed a different pattern, with slowest response times recorded for expressions of moderate intensity. Overall, responses were slowest, but also most accurate, for female faces. Relative to male observers, women showed greater accuracy and speed when recognizing female expressions. Dwell time analyses revealed that attention to the eyes was about three times greater than on the mouth, with fearful eyes in particular attracting longer dwell times. The mouth region was attended to the most for fearful, angry, and disgusted expressions and least for surprise. These results extend upon previous findings to show important effects of expression, emotion intensity, and sex on expression recognition and gaze behaviour, and may have implications for understanding the ways in which emotion recognition abilities break down.

  11. Identification of Emotional Facial Expressions: Effects of Expression, Intensity, and Sex on Eye Gaze.

    Science.gov (United States)

    Wells, Laura Jean; Gillespie, Steven Mark; Rotshtein, Pia

    2016-01-01

    The identification of emotional expressions is vital for social interaction, and can be affected by various factors, including the expressed emotion, the intensity of the expression, the sex of the face, and the gender of the observer. This study investigates how these factors affect the speed and accuracy of expression recognition, as well as dwell time on the two most significant areas of the face: the eyes and the mouth. Participants were asked to identify expressions from female and male faces displaying six expressions (anger, disgust, fear, happiness, sadness, and surprise), each with three levels of intensity (low, moderate, and normal). Overall, responses were fastest and most accurate for happy expressions, but slowest and least accurate for fearful expressions. More intense expressions were also classified most accurately. Reaction time showed a different pattern, with slowest response times recorded for expressions of moderate intensity. Overall, responses were slowest, but also most accurate, for female faces. Relative to male observers, women showed greater accuracy and speed when recognizing female expressions. Dwell time analyses revealed that attention to the eyes was about three times greater than on the mouth, with fearful eyes in particular attracting longer dwell times. The mouth region was attended to the most for fearful, angry, and disgusted expressions and least for surprise. These results extend upon previous findings to show important effects of expression, emotion intensity, and sex on expression recognition and gaze behaviour, and may have implications for understanding the ways in which emotion recognition abilities break down.

  12. Profiling Gene Expression in Germinating Brassica Roots.

    Science.gov (United States)

    Park, Myoung Ryoul; Wang, Yi-Hong; Hasenstein, Karl H

    2014-01-01

    Based on previously developed solid-phase gene extraction (SPGE) we examined the mRNA profile in primary roots of Brassica rapa seedlings for highly expressed genes like ACT7 (actin7), TUB (tubulin1), UBQ (ubiquitin), and low expressed GLK (glucokinase) during the first day post-germination. The assessment was based on the mRNA load of the SPGE probe of about 2.1 ng. The number of copies of the investigated genes changed spatially along the length of primary roots. The expression level of all genes differed significantly at each sample position. Among the examined genes ACT7 expression was most even along the root. UBQ was highest at the tip and root-shoot junction (RS). TUB and GLK showed a basipetal gradient. The temporal expression of UBQ was highest in the MZ 9 h after primary root emergence and higher than at any other sample position. Expressions of GLK in EZ and RS increased gradually over time. SPGE extraction is the result of oligo-dT and oligo-dA hybridization and the results illustrate that SPGE can be used for gene expression profiling at high spatial and temporal resolution. SPGE needles can be used within two weeks when stored at 4 °C. Our data indicate that gene expression studies that are based on the entire root miss important differences in gene expression that SPGE is able to resolve for example growth adjustments during gravitropism.

  13. TRPM4 protein expression in prostate cancer

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Soldini, Davide; Jung, Maria

    2016-01-01

    BACKGROUND: Transient receptor potential cation channel, subfamily M, member 4 (TRPM4) messenger RNA (mRNA) has been shown to be upregulated in prostate cancer (PCa) and might be a new promising tissue biomarker. We evaluated TRPM4 protein expression and correlated the expression level.......79-2.62; p = 0.01-0.03 for the two observers) when compared to patients with a lower staining intensity. CONCLUSIONS: TRPM4 protein expression is widely expressed in benign and cancerous prostate tissue, with highest staining intensities found in PCa. Overexpression of TRPM4 in PCa (combination of high...

  14. Automatic emotional expression analysis from eye area

    Science.gov (United States)

    Akkoç, Betül; Arslan, Ahmet

    2015-02-01

    Eyes play an important role in expressing emotions in nonverbal communication. In the present study, emotional expression classification was performed based on the features that were automatically extracted from the eye area. Fırst, the face area and the eye area were automatically extracted from the captured image. Afterwards, the parameters to be used for the analysis through discrete wavelet transformation were obtained from the eye area. Using these parameters, emotional expression analysis was performed through artificial intelligence techniques. As the result of the experimental studies, 6 universal emotions consisting of expressions of happiness, sadness, surprise, disgust, anger and fear were classified at a success rate of 84% using artificial neural networks.

  15. Modulation of gene expression made easy

    DEFF Research Database (Denmark)

    Solem, Christian; Jensen, Peter Ruhdal

    2002-01-01

    A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example...... that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the las operon, containing the genes pfk, pyk, and ldh, was modulated by integrating a truncated copy of the pfk gene. Importantly, the modulation affected...

  16. Judgments of subtle facial expressions of emotion.

    Science.gov (United States)

    Matsumoto, David; Hwang, Hyisung C

    2014-04-01

    Most studies on judgments of facial expressions of emotion have primarily utilized prototypical, high-intensity expressions. This paper examines judgments of subtle facial expressions of emotion, including not only low-intensity versions of full-face prototypes but also variants of those prototypes. A dynamic paradigm was used in which observers were shown a neutral expression followed by the target expression to judge, and then the neutral expression again, allowing for a simulation of the emergence of the expression from and then return to a baseline. We also examined how signal and intensity clarities of the expressions (explained more fully in the Introduction) were associated with judgment agreement levels. Low-intensity, full-face prototypical expressions of emotion were judged as the intended emotion at rates significantly greater than chance. A number of the proposed variants were also judged as the intended emotions. Both signal and intensity clarities were individually associated with agreement rates; when their interrelationships were taken into account, signal clarity independently predicted agreement rates but intensity clarity did not. The presence or absence of specific muscles appeared to be more important to agreement rates than their intensity levels, with the exception of the intensity of zygomatic major, which was positively correlated with agreement rates for judgments of joy.

  17. Examining emotional expressions in discourse: methodological considerations

    Science.gov (United States)

    Hufnagel, Elizabeth; Kelly, Gregory J.

    2017-10-01

    This methodological paper presents an approach for examining emotional expressions through discourse analysis and ethnographic methods. Drawing on trends in the current literature in science education, we briefly explain the importance of emotions in science education and examine the current research methodologies used in interactional emotion studies. We put forth and substantiate a methodological approach that attends to the interactional, contextual, intertextual, and consequential aspects of emotional expressions. By examining emotional expressions in the discourse in which they are constructed, emotional expressions are identified through semantics, contextualization, and linguistic features. These features make salient four dimensions of emotional expressions: aboutness, frequency, type, and ownership. Drawing on data from a large empirical study of pre-service elementary teachers' emotional expressions about climate change in a science course, we provide illustrative examples to describe what counts as emotional expressions in situ. In doing so we explain how our approach makes salient the nuanced nature of such expressions as well as the broader discourse in which they are constructed and the implications for researching emotional expressions in science education discourse. We suggest reasons why this discourse orientated research methodology can contribute to the interactional study of emotions in science education contexts.

  18. Social Use of Facial Expressions in Hylobatids

    Science.gov (United States)

    Scheider, Linda; Waller, Bridget M.; Oña, Leonardo; Burrows, Anne M.; Liebal, Katja

    2016-01-01

    Non-human primates use various communicative means in interactions with others. While primate gestures are commonly considered to be intentionally and flexibly used signals, facial expressions are often referred to as inflexible, automatic expressions of affective internal states. To explore whether and how non-human primates use facial expressions in specific communicative interactions, we studied five species of small apes (gibbons) by employing a newly established Facial Action Coding System for hylobatid species (GibbonFACS). We found that, despite individuals often being in close proximity to each other, in social (as opposed to non-social contexts) the duration of facial expressions was significantly longer when gibbons were facing another individual compared to non-facing situations. Social contexts included grooming, agonistic interactions and play, whereas non-social contexts included resting and self-grooming. Additionally, gibbons used facial expressions while facing another individual more often in social contexts than non-social contexts where facial expressions were produced regardless of the attentional state of the partner. Also, facial expressions were more likely ‘responded to’ by the partner’s facial expressions when facing another individual than non-facing. Taken together, our results indicate that gibbons use their facial expressions differentially depending on the social context and are able to use them in a directed way in communicative interactions with other conspecifics. PMID:26978660

  19. Clusterin expression in elastofibroma dorsi.

    Science.gov (United States)

    Aigelsreiter, Ariane; Pichler, Martin; Pixner, Thomas; Janig, Elke; Schuller, Monika; Lackner, Carolin; Scheipl, Susanne; Beham, Alfred; Regauer, Sigrid

    2013-05-01

    Elastofibroma dorsi is a benign soft tissue lesion composed of abnormal elastic fibers. Degenerated elastic fibers in skin and liver are associated with clusterin, an apoprotein that shares functional properties with small heat shock proteins. We evaluated the staining pattern and possible role of clusterin in elastofibroma dorsi. Twenty-one subcutaneous elastofibromas from the scapular region were evaluated with Elastica van Gieson and Orcein stains, immunohistochemically with antibodies to clusterin, smooth muscle actin, S-100, vimentin and CD34 and correlated with clinical data with respect to physical trauma. Clusterin correlated with the staining pattern of Elastica van Gieson and labelled abnormal broad coarse fibrillar and globular elastic fibers in all elastofibromas. Orcein stains additionally identified fine oxytalan fibers which were not stained by clusterin. Clusterin staining was observed only on the outside of the elastin fibers, while the cores of fibers and globules were unstained. 4/21 elastofibromas showed cellular nodules with a myxoid/collagenous stroma. The round to oval cells showed cytoplasmic staining with vimentin and clusterin; CD34 labelled mostly cell membranes. The cells lacked SMA and S-100 expression. The central areas of the nodules were devoid of elastic fibers, but the periphery contained coarse fibers and globules. 9/ 11 patients, for whom clinical data were available, reported trauma to the scapular region. Many investigated ED were associated with trauma, which supports a reactive/degenerative etiology of ED. The abnormal large elastic fibers in all ED were enveloped by clusterin. Clusterin deposition may protect elastic fibers from degradation and thus contribute indirectly to the tumor-like presentation of ED.

  20. Gene expression inference with deep learning.

    Science.gov (United States)

    Chen, Yifei; Li, Yi; Narayan, Rajiv; Subramanian, Aravind; Xie, Xiaohui

    2016-06-15

    Large-scale gene expression profiling has been widely used to characterize cellular states in response to various disease conditions, genetic perturbations, etc. Although the cost of whole-genome expression profiles has been dropping steadily, generating a compendium of expression profiling over thousands of samples is still very expensive. Recognizing that gene expressions are often highly correlated, researchers from the NIH LINCS program have developed a cost-effective strategy of profiling only ∼1000 carefully selected landmark genes and relying on computational methods to infer the expression of remaining target genes. However, the computational approach adopted by the LINCS program is currently based on linear regression (LR), limiting its accuracy since it does not capture complex nonlinear relationship between expressions of genes. We present a deep learning method (abbreviated as D-GEX) to infer the expression of target genes from the expression of landmark genes. We used the microarray-based Gene Expression Omnibus dataset, consisting of 111K expression profiles, to train our model and compare its performance to those from other methods. In terms of mean absolute error averaged across all genes, deep learning significantly outperforms LR with 15.33% relative improvement. A gene-wise comparative analysis shows that deep learning achieves lower error than LR in 99.97% of the target genes. We also tested the performance of our learned model on an independent RNA-Seq-based GTEx dataset, which consists of 2921 expression profiles. Deep learning still outperforms LR with 6.57% relative improvement, and achieves lower error in 81.31% of the target genes. D-GEX is available at https://github.com/uci-cbcl/D-GEX CONTACT: xhx@ics.uci.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Expression of CD133 in acute leukemia.

    Science.gov (United States)

    Tolba, Fetnat M; Foda, Mona E; Kamal, Howyda M; Elshabrawy, Deena A

    2013-06-01

    There have been conflicting results regarding a correlation between CD133 expression and disease outcome. To assess CD133 expression in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and to evaluate its correlation with the different clinical and laboratory data as well as its relation to disease outcome, the present study included 60 newly diagnosed acute leukemic patients; 30 ALL patients with a male to female ratio of 1.5:1 and their ages ranged from 9 months to 48 years, and 30 AML patients with a male to female ratio of 1:1 and their ages ranged from 17 to 66 years. Flow cytometric assessment of CD133 expression was performed on blast cells. In ALL, no correlations were elicited between CD133 expression and some monoclonal antibodies, but in AML group, there was a significant positive correlation between CD133 and HLA-DR, CD3, CD7 and TDT, CD13 and CD34. In ALL group, patients with negative CD133 expression achieved complete remission more than patients with positive CD133 expression. In AML group, there was no statistically significant association found between positive CD133 expression and treatment outcome. The Kaplan-Meier curve illustrated a high significant negative correlation between CD133 expression and the overall survival of the AML patients. CD133 expression is an independent prognostic factor in acute leukemia, especially ALL patients and its expression could characterize a group of acute leukemic patients with higher resistance to standard chemotherapy and relapse. CD133 expression was highly associated with poor prognosis in acute leukemic patients.

  2. Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells

    International Nuclear Information System (INIS)

    Qin Zhaoling; Zhao Ping; Zhang Xiaolian; Yu Jianguo; Cao Mingmei; Zhao Lanjuan; Luan Jie; Qi Zhongtian

    2004-01-01

    Two candidate small interfering RNAs (siRNAs) corresponding to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike gene were designed and in vitro transcribed to explore the possibility of silencing SARS-CoV S gene. The plasmid pEGFP-optS, which contains the codon-optimized SARS-CoV S gene and expresses spike-EGFP fusion protein (S-EGFP) as silencing target and expressing reporter, was transfected with siRNAs into HEK 293T cells. At various time points of posttransfection, the levels of S-EGFP expression and amounts of spike mRNA transcript were detected by fluorescence microscopy, flow cytometry, Western blot, and real-time quantitative PCR, respectively. The results showed that the cells transfected with pEGFP-optS expressed S-EGFP fusion protein at a higher level compared with those transfected with pEGFP-S, which contains wildtype SARS-CoV spike gene sequence. The green fluorescence, mean fluorescence intensity, and SARS-CoV S RNA transcripts were found significantly reduced, and the expression of SARS-CoV S glycoprotein was strongly inhibited in those cells co-transfected with either EGFP- or S-specific siRNAs. Our findings demonstrated that the S-specific siRNAs used in this study were able to specifically and effectively inhibit SARS-CoV S glycoprotein expression in cultured cells through blocking the accumulation of S mRNA, which may provide an approach for studies on the functions of SARS-CoV S gene and development of novel prophylactic or therapeutic agents for SARS-CoV

  3. Ultrasound-targeted microbubble destruction enhances naked plasmid DNA transfection in rabbit Achilles tendons in vivo.

    Science.gov (United States)

    Qiu, L; Zhang, L; Wang, L; Jiang, Y; Luo, Y; Peng, Y; Lin, L

    2012-07-01

    The study was to investigate the probability of increasing the transfection of the gene in tendons by ultrasound-targeted microbubble destruction (UTMD), and to search for the most suitable transfection conditions. A mixture of microbubbles and enhanced green fluorescent protein (EGFP) plasmids was injected into rabbit Achilles tendons by different administration routes and the tendons were ultrasound pulse by different ultrasonic conditions in order to determine the most appropriate conditions. Then, the rabbits were divided into four groups: (1) ultrasound + microbubbles + plasmid; (2) ultrasound+ plasmid; (3) microbubble + plasmid; (4) plasmid only. EGFP expression in the tendons and other tissues, and the damage to tendon and paratenon were all observed. The results showed that EGFP expression in the tendon was higher by ultrasound pulse with 2 W cm(-2) of output intensity and a 20% duty cycle for 10 min. Local injection was determined to be the better administration route. Among the four groups, EGFP expression in Group 1 was higher than that in other groups. EGFP expression was highest on seventh day, then it gradually decrease over time, and lasted more than 56 days. EGFP expression was not found in other tissues. There was no obvious injury caused by UTMD. Under suitable conditions, it is feasible to use UTMD as a safe and effective gene transfection therapy for tendon injuries.

  4. Foxp3 expression in human cancer cells

    Directory of Open Access Journals (Sweden)

    Gourgoulianis Konstantinos I

    2008-04-01

    Full Text Available Abstract Objective Transcription factor forkhead box protein 3 (Foxp3 specifically characterizes the thymically derived naturally occurring regulatory T cells (Tregs. Limited evidence indicates that it is also expressed, albeit to a lesser extent, in tissues other than thymus and spleen, while, very recently, it was shown that Foxp3 is expressed by pancreatic carcinoma. This study was scheduled to investigate whether expression of Foxp3 transcripts and mature protein occurs constitutively in various tumor types. Materials and methods Twenty five tumor cell lines of different tissue origins (lung cancer, colon cancer, breast cancer, melanoma, erythroid leukemia, acute T-cell leukemia were studied. Detection of Foxp3 mRNA was performed using both conventional RT-PCR and quantitative real-time PCR while protein expression was assessed by immunocytochemistry and flow cytometry, using different antibody clones. Results Foxp3 mRNA as well as Foxp3 protein was detected in all tumor cell lines, albeit in variable levels, not related to the tissue of origin. This expression correlated with the expression levels of IL-10 and TGFb1. Conclusion We offer evidence that Foxp3 expression, characterizes tumor cells of various tissue origins. The biological significance of these findings warrants further investigation in the context of tumor immune escape, and especially under the light of current anti-cancer efforts interfering with Foxp3 expression.

  5. Explanations for Contempt Expressed Towards Old People.

    Science.gov (United States)

    Maxwell, Eleanor Krassen; Maxwell, Robert J.

    The issue of contempt expressed towards the aged was examined from a cross-cultural perspective. Eight reasons for expressions of contempt emerged from a study of 95 societies drawn from the Standard Cross-Cultural Sample, and were treated as independent variables, with the overall level of contempt as the dependent variable, in a application of…

  6. Equilibrium-constant expressions for aqueous plutonium

    International Nuclear Information System (INIS)

    Silver, G.L.

    2010-01-01

    Equilibrium-constant expressions for Pu disproportionation reactions traditionally contain three or four terms representing the concentrations or fractions of the oxidation states. The expressions can be rewritten so that one of the oxidation states is replaced by a term containing the oxidation number of the plutonium. Experimental estimations of the numerical values of the constants can then be checked in several ways. (author)

  7. Behavior, Experience and Expression: Some Research Considerations.

    Science.gov (United States)

    Romanyshyn, Robert D.

    Utilizing research conducted on nostalgia, this paper shows how a phenomenological approach assists in understanding behavior, experience and expression. Moreover, a clearer understanding of them aids one's research with and comprehension of nostalgia. Human action can be studied from the experiential, behavioral and expressive perspectives. These…

  8. Serial analysis of gene expression (SAGE)

    NARCIS (Netherlands)

    van Ruissen, Fred; Baas, Frank

    2007-01-01

    In 1995, serial analysis of gene expression (SAGE) was developed as a versatile tool for gene expression studies. SAGE technology does not require pre-existing knowledge of the genome that is being examined and therefore SAGE can be applied to many different model systems. In this chapter, the SAGE

  9. Dihydrotestostenone increase the gene expression of androgen ...

    African Journals Online (AJOL)

    HNTEP cells were grown in basal medium and treated with DHT in different conditions. HNTEP cells under treatment with DHT (10-13 M) induced an increase in FHL-2 expression. In turn, high DHT concentrations (10-8 M) induced an increase in the expression SHP-1. The present data suggest that the SHP-1 and FHL-2 ...

  10. Predictable tuning of protein expression in bacteria

    DEFF Research Database (Denmark)

    Bonde, Mads; Pedersen, Margit; Klausen, Michael Schantz

    2016-01-01

    We comprehensively assessed the contribution of the Shine-Dalgarno sequence to protein expression and used the data to develop EMOPEC (Empirical Model and Oligos for Protein Expression Changes; http://emopec.biosustain.dtu.dk). EMOPEC is a free tool that makes it possible to modulate the expressi...

  11. A Robot with Complex Facial Expressions

    Directory of Open Access Journals (Sweden)

    J. Takeno

    2009-08-01

    Full Text Available The authors believe that the consciousness of humans basically originates from languages and their association-like flow of consciousness, and that feelings are generated accompanying respective languages. We incorporated artificial consciousness into a robot; achieved an association flow of language like flow of consciousness; and developed a robot called Kansei that expresses its feelings according to the associations occurring in the robot. To be able to fully communicate with humans, robots must be able to display complex expressions, such as a sense of being thrilled. We therefore added to the Kansei robot a device to express complex feelings through its facial expressions. The Kansei robot is actually an artificial skull made of aluminum, with servomotors built into it. The face is made of relatively soft polyethylene, which is formed to appear like a human face. Facial expressions are generated using 19 servomotors built into the skull, which pull metal wires attached to the facial “skin” to create expressions. The robot at present is capable of making six basic expressions as well as complex expressions, such as happiness and fear combined.

  12. Automating InDesign with Regular Expressions

    CERN Document Server

    Kahrel, Peter

    2006-01-01

    If you need to make automated changes to InDesign documents beyond what basic search and replace can handle, you need regular expressions, and a bit of scripting to make them work. This Short Cut explains both how to write regular expressions, so you can find and replace the right things, and how to use them in InDesign specifically.

  13. Construction of expression vectors carrying mouse peroxisomal ...

    African Journals Online (AJOL)

    The aim of this study was to construct expression vectors carrying mouse peroxisomal protein gene (PEP-cDNA) in prokaryotic and mammalian expression vectors in ... pGEX6p2-PEP and pUcD3-FLAG-PEP constructed vectors were transformed into the one shot TOP10 and JM105 bacterial competent cells, respectively.

  14. Assessment of Outcomes of Free Expression Courses.

    Science.gov (United States)

    Andsager, Julie; Ross, Susan Dente

    1999-01-01

    Assesses outcomes of instruction in three college-senior-level courses on freedom of expression. Suggests that increased attention to freedom-of-expression issues may have resulted in broader understanding of First Amendment issues, and individual and media rights. Notes that students seem to develop an appreciation of the reflexive nature of…

  15. Local expressions for one-loop calculations

    International Nuclear Information System (INIS)

    Wasson, D.A.; Koonin, S.E.

    1991-01-01

    We develop local expressions for the contributions of the short-wavelength vacuum modes to the one-loop vacuum energy. These expressions significantly improve the convergence properties of various ''brute-force'' calculational methods. They also provide a continuous series of approximations that interpolate between the brute-force calculations and the derivative expansion

  16. Artistic Expression: Another Challenge for Rural Education

    Directory of Open Access Journals (Sweden)

    Sonia Álvarez-Castro

    2012-12-01

    Full Text Available This paper identifies the parameters required to create opportunities that would strengthen the social fabric and would promote a comprehensive development through the artistic expression as a method for expressing feelings and constructing –cultural and social– identities as individuals, which, in our global context have been eroded by the homogenization of experiences.

  17. Dirichlet expression for L(1, χ )

    Indian Academy of Sciences (India)

    We show that this expression with obvious modification is valid for the general primitive Dirichlet character χ. Keywords. Hurwitz zeta function; Dirichlet character; Dirichlet L-series; primitive character. 1. Introduction. In Dirichlet's famous work dealing with class number formula, the value of L(1,χ) is expressed in terms of finite ...

  18. Sad Facial Expressions Increase Choice Blindness

    Directory of Open Access Journals (Sweden)

    Yajie Wang

    2018-01-01

    Full Text Available Previous studies have discovered a fascinating phenomenon known as choice blindness—individuals fail to detect mismatches between the face they choose and the face replaced by the experimenter. Although previous studies have reported a couple of factors that can modulate the magnitude of choice blindness, the potential effect of facial expression on choice blindness has not yet been explored. Using faces with sad and neutral expressions (Experiment 1 and faces with happy and neutral expressions (Experiment 2 in the classic choice blindness paradigm, the present study investigated the effects of facial expressions on choice blindness. The results showed that the detection rate was significantly lower on sad faces than neutral faces, whereas no significant difference was observed between happy faces and neutral faces. The exploratory analysis of verbal reports found that participants who reported less facial features for sad (as compared to neutral expressions also tended to show a lower detection rate of sad (as compared to neutral faces. These findings indicated that sad facial expressions increased choice blindness, which might have resulted from inhibition of further processing of the detailed facial features by the less attractive sad expressions (as compared to neutral expressions.

  19. Sad Facial Expressions Increase Choice Blindness.

    Science.gov (United States)

    Wang, Yajie; Zhao, Song; Zhang, Zhijie; Feng, Wenfeng

    2017-01-01

    Previous studies have discovered a fascinating phenomenon known as choice blindness-individuals fail to detect mismatches between the face they choose and the face replaced by the experimenter. Although previous studies have reported a couple of factors that can modulate the magnitude of choice blindness, the potential effect of facial expression on choice blindness has not yet been explored. Using faces with sad and neutral expressions (Experiment 1) and faces with happy and neutral expressions (Experiment 2) in the classic choice blindness paradigm, the present study investigated the effects of facial expressions on choice blindness. The results showed that the detection rate was significantly lower on sad faces than neutral faces, whereas no significant difference was observed between happy faces and neutral faces. The exploratory analysis of verbal reports found that participants who reported less facial features for sad (as compared to neutral) expressions also tended to show a lower detection rate of sad (as compared to neutral) faces. These findings indicated that sad facial expressions increased choice blindness, which might have resulted from inhibition of further processing of the detailed facial features by the less attractive sad expressions (as compared to neutral expressions).

  20. Biased Facial Expression Interpretation in Shy Children

    Science.gov (United States)

    Kokin, Jessica; Younger, Alastair; Gosselin, Pierre; Vaillancourt, Tracy

    2016-01-01

    The relationship between shyness and the interpretations of the facial expressions of others was examined in a sample of 123 children aged 12 to 14?years. Participants viewed faces displaying happiness, fear, anger, disgust, sadness, surprise, as well as a neutral expression, presented on a computer screen. The children identified each expression…

  1. Characterization of differentially expressed genes using high-dimensional co-expression networks

    DEFF Research Database (Denmark)

    Coelho Goncalves de Abreu, Gabriel; Labouriau, Rodrigo S.

    2010-01-01

    We present a technique to characterize differentially expressed genes in terms of their position in a high-dimensional co-expression network. The set-up of Gaussian graphical models is used to construct representations of the co-expression network in such a way that redundancy and the propagation...... that allow to make effective inference in problems with high degree of complexity (e.g. several thousands of genes) and small number of observations (e.g. 10-100) as typically occurs in high throughput gene expression studies. Taking advantage of the internal structure of decomposable graphical models, we...... construct a compact representation of the co-expression network that allows to identify the regions with high concentration of differentially expressed genes. It is argued that differentially expressed genes located in highly interconnected regions of the co-expression network are less informative than...

  2. Polycistronic gene expression in Aspergillus niger.

    Science.gov (United States)

    Schuetze, Tabea; Meyer, Vera

    2017-09-25

    Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. The P2A peptide can be used to express at

  3. Expression of Sox genes in tooth development.

    Science.gov (United States)

    Kawasaki, Katsushige; Kawasaki, Maiko; Watanabe, Momoko; Idrus, Erik; Nagai, Takahiro; Oommen, Shelly; Maeda, Takeyasu; Hagiwara, Nobuko; Que, Jianwen; Sharpe, Paul T; Ohazama, Atsushi

    2015-01-01

    Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development.

  4. Emotion Expression of Robot with Personality

    Directory of Open Access Journals (Sweden)

    Xue Hu

    2013-01-01

    Full Text Available A robot emotional expression model based on Hidden Markov Model (HMM is built to enable robots which have different personalities to response in a more satisfactory emotional level. Gross emotion regulation theory and Five Factors Model (FFM which are the theoretical basis are firstly described. And then the importance of the personality effect on the emotion expression process is proposed, and how to make the effect quantization is discussed. After that, the algorithm of HMM is used to describe the process of emotional state transition and expression, and the performance transferring probability affected by personality is calculated. At last, the algorithm model is simulated and applied in a robot platform. The results prove that the emotional expression model can acquire humanlike expressions and improve the human-computer interaction.

  5. Willingness to express emotions to caregiving spouses.

    Science.gov (United States)

    Monin, Joan K; Martire, Lynn M; Schulz, Richard; Clark, Margaret S

    2009-02-01

    This study examined the association between care-recipients' willingness to express emotions to spousal caregivers and caregiver's well-being and support behaviors. Using self-report measures in the context of a larger study, 262 care-recipients with osteoarthritis reported on their willingness to express emotions to caregivers, and caregivers reported on their stress and insensitive responding to care-recipients. Results revealed that care-recipients' willingness to express happiness was associated with less insensitive caregiver responding, and willingness to express interpersonal emotions (e.g., compassion, guilt) was associated with less caregiving stress. There were also gender differences, such that caregiving wives, in particular, benefited from their husband's willingness to express vulnerable (e.g., anxiety, sadness) and interpersonal emotions. (c) 2009 APA, all rights reserved

  6. Personality expression in Chinese language use.

    Science.gov (United States)

    Qiu, Lin; Lu, Jiahui; Ramsay, Jonathan; Yang, Shanshan; Qu, Weina; Zhu, Tingshao

    2017-12-01

    To date, little research has investigated personality expressions in languages other than English. Given that the Chinese language has the largest number of native speakers in the world, it is vitally important to examine the associations between personality and Chinese language use. In this research, we analysed Chinese microblogs and identified word categories and factorial structures associated with personality traits. We also compared our results with previous findings in English and showed that linguistic expression of personality has both universal- and language-specific aspects. Expression of personality via content words is more likely to be consistent across languages than expression via function words. This makes an important step towards uncovering universal patterns of personality expression in language. © 2016 International Union of Psychological Science.

  7. PRAME gene expression profile in medulloblastoma

    Directory of Open Access Journals (Sweden)

    Tânia Maria Vulcani-Freitas

    2011-02-01

    Full Text Available Medulloblastoma is the most common malignant tumors of central nervous system in the childhood. The treatment is severe, harmful and, thus, has a dismal prognosis. As PRAME is present in various cancers, including meduloblastoma, and has limited expression in normal tissues, this antigen can be an ideal vaccine target for tumor immunotherapy. In order to find a potential molecular target, we investigated PRAME expression in medulloblastoma fragments and we compare the results with the clinical features of each patient. Analysis of gene expression was performed by real-time quantitative PCR from 37 tumor samples. The Mann-Whitney test was used to analysis the relationship between gene expression and clinical characteristics. Kaplan-Meier curves were used to evaluate survival. PRAME was overexpressed in 84% samples. But no statistical association was found between clinical features and PRAME overexpression. Despite that PRAME gene could be a strong candidate for immunotherapy since it is highly expressed in medulloblastomas.

  8. Development and evaluation of novel recombinant adenovirus-based vaccine candidates for infectious bronchitis virus and Mycoplasma gallisepticum in chickens.

    Science.gov (United States)

    Zhang, Dongchao; Long, Yuqing; Li, Meng; Gong, Jianfang; Li, Xiaohui; Lin, Jing; Meng, Jiali; Gao, Keke; Zhao, Ruili; Jin, Tianming

    2018-04-01

    Avian infectious bronchitis caused by the infectious bronchitis virus (IBV), and mycoplasmosis caused by Mycoplasma gallisepticum (MG) are two major respiratory diseases in chickens that have resulted in severe economic losses in the poultry industry. We constructed a recombinant adenovirus that simultaneously expresses the S1 spike glycoprotein of IBV and the TM-1 protein of MG (pBH-S1-TM-1-EGFP). For comparison, we constructed two recombinant adenoviruses (pBH-S1-EGFP and pBH-TM-1-EGFP) that express either the S1 spike glycoprotein or the TM-1 protein alone. The protective efficacy of these three vaccine constructs against challenge with IBV and/or MG was evaluated in specific pathogen free chickens. Groups of seven-day-old specific pathogen free chicks were immunized twice, two weeks apart, via the oculonasal route with the pBH-S1-TM-1-EGFP, pBH-S1-EGFP, or pBH-TM-1-EGFP vaccine candidates or the commercial attenuated infectious bronchitis vaccine strain H52 and MG vaccine strain F-36 (positive controls), and challenged with virulent IBV or MG two weeks later. Interestingly, by days 7 and 14 after the booster immunization, pBH-S1-TM-1-EGFP-induced antibody titre was significantly higher (P attenuated commercial IBV vaccine; however, there was no significant difference between the pBH-S1-TM-1-EGFP and attenuated commercial MG vaccine groups (P > 0.05). The clinical signs, the gross, and histopathological lesions scores of the adenovirus vaccine constructs were not significantly different from that of the attenuated commercial IBV or MG vaccines (positive controls) (P > 0.05). These results demonstrate the potential of the bivalent pBH-S1-TM-1-EGFP adenovirus construct as a combination vaccine against IB and mycoplasmosis.

  9. Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data

    OpenAIRE

    Ezer, Daphne; Moignard, Victoria; G?ttgens, Berthold; Adryan, Boris

    2016-01-01

    Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete ...

  10. The MPI facial expression database--a validated database of emotional and conversational facial expressions.

    Directory of Open Access Journals (Sweden)

    Kathrin Kaulard

    Full Text Available The ability to communicate is one of the core aspects of human life. For this, we use not only verbal but also nonverbal signals of remarkable complexity. Among the latter, facial expressions belong to the most important information channels. Despite the large variety of facial expressions we use in daily life, research on facial expressions has so far mostly focused on the emotional aspect. Consequently, most databases of facial expressions available to the research community also include only emotional expressions, neglecting the largely unexplored aspect of conversational expressions. To fill this gap, we present the MPI facial expression database, which contains a large variety of natural emotional and conversational expressions. The database contains 55 different facial expressions performed by 19 German participants. Expressions were elicited with the help of a method-acting protocol, which guarantees both well-defined and natural facial expressions. The method-acting protocol was based on every-day scenarios, which are used to define the necessary context information for each expression. All facial expressions are available in three repetitions, in two intensities, as well as from three different camera angles. A detailed frame annotation is provided, from which a dynamic and a static version of the database have been created. In addition to describing the database in detail, we also present the results of an experiment with two conditions that serve to validate the context scenarios as well as the naturalness and recognizability of the video sequences. Our results provide clear evidence that conversational expressions can be recognized surprisingly well from visual information alone. The MPI facial expression database will enable researchers from different research fields (including the perceptual and cognitive sciences, but also affective computing, as well as computer vision to investigate the processing of a wider range of natural

  11. The MPI Facial Expression Database — A Validated Database of Emotional and Conversational Facial Expressions

    Science.gov (United States)

    Kaulard, Kathrin; Cunningham, Douglas W.; Bülthoff, Heinrich H.; Wallraven, Christian

    2012-01-01

    The ability to communicate is one of the core aspects of human life. For this, we use not only verbal but also nonverbal signals of remarkable complexity. Among the latter, facial expressions belong to the most important information channels. Despite the large variety of facial expressions we use in daily life, research on facial expressions has so far mostly focused on the emotional aspect. Consequently, most databases of facial expressions available to the research community also include only emotional expressions, neglecting the largely unexplored aspect of conversational expressions. To fill this gap, we present the MPI facial expression database, which contains a large variety of natural emotional and conversational expressions. The database contains 55 different facial expressions performed by 19 German participants. Expressions were elicited with the help of a method-acting protocol, which guarantees both well-defined and natural facial expressions. The method-acting protocol was based on every-day scenarios, which are used to define the necessary context information for each expression. All facial expressions are available in three repetitions, in two intensities, as well as from three different camera angles. A detailed frame annotation is provided, from which a dynamic and a static version of the database have been created. In addition to describing the database in detail, we also present the results of an experiment with two conditions that serve to validate the context scenarios as well as the naturalness and recognizability of the video sequences. Our results provide clear evidence that conversational expressions can be recognized surprisingly well from visual information alone. The MPI facial expression database will enable researchers from different research fields (including the perceptual and cognitive sciences, but also affective computing, as well as computer vision) to investigate the processing of a wider range of natural facial expressions

  12. Vitamin D Receptor Expression in Dogs

    Science.gov (United States)

    Gow, A.G.; Milne, E.; Drummond, D.; Smith, S.; Handel, I.; Mellanby, R.J.

    2018-01-01

    Background There is growing evidence linking low blood vitamin D concentration to numerous diseases in people and in dogs. Vitamin D influences cellular function by signaling through the vitamin D receptor (VDR). Little is known about which non‐skeletal tissues express the VDR or how inflammation influences its expression in the dog. Objectives To define which non‐skeletal canine tissues express the VDR and to investigate expression in inflamed small intestine. Animals Thirteen non‐skeletal tissues were collected prospectively from 6 control dogs. Thirty‐five dogs diagnosed with a chronic enteropathy (CE) and 24 control dogs were prospectively enrolled and duodenal biopsies were evaluated for VDR expression. Methods Prospective; blinded assessment of canine intestinal VDR. Dogs with CE were included once other identifiable causes of intestinal disease were excluded. Age matched controls were included with no intestinal clinical signs. VDR expression was assessed immunohistochemically in all samples, using a Rat IgG VDR monoclonal antibody. Quantitative real‐time polymerase chain reaction (qPCR) was also used for duodenal biopsies. Results VDR expression as assessed by immunohistochemistry (IHC) was highest in the kidney, duodenum, skin, ileum and spleen, and weak in the colon, heart, lymph node, liver, lung, and ovary. Gastric and testicular tissue did not express the VDR. There was no statistical difference in duodenal VDR expression between the 24 healthy dogs and 34 dogs with CE when quantified by either qPCR (P = 0.87) or IHC (P = 0.099). Conclusions and Clinical Importance The lack of down regulation of VDR expression in inflamed intestine contrasts with previous studies in humans. Our findings support future studies to investigate whether vitamin D and its analogues can be used to modulate intestinal inflammation in the dog. PMID:29469965

  13. The functional landscape of mouse gene expression

    Directory of Open Access Journals (Sweden)

    Zhang Wen

    2004-12-01

    Full Text Available Abstract Background Large-scale quantitative analysis of transcriptional co-expression has been used to dissect regulatory networks and to predict the functions of new genes discovered by genome sequencing in model organisms such as yeast. Although the idea that tissue-specific expression is indicative of gene function in mammals is widely accepted, it has not been objectively tested nor compared with the related but distinct strategy of correlating gene co-expression as a means to predict gene function. Results We generated microarray expression data for nearly 40,000 known and predicted mRNAs in 55 mouse tissues, using custom-built oligonucleotide arrays. We show that quantitative transcriptional co-expression is a powerful predictor of gene function. Hundreds of functional categories, as defined by Gene Ontology 'Biological Processes', are associated with characteristic expression patterns across all tissues, including categories that bear no overt relationship to the tissue of origin. In contrast, simple tissue-specific restriction of expression is a poor predictor of which genes are in which functional categories. As an example, the highly conserved mouse gene PWP1 is widely expressed across different tissues but is co-expressed with many RNA-processing genes; we show that the uncharacterized yeast homolog of PWP1 is required for rRNA biogenesis. Conclusions We conclude that 'functional genomics' strategies based on quantitative transcriptional co-expression will be as fruitful in mammals as they have been in simpler organisms, and that transcriptional control of mammalian physiology is more modular than is generally appreciated. Our data and analyses provide a public resource for mammalian functional genomics.

  14. Simultaneous Breast Expression in Breastfeeding Women Is More Efficacious Than Sequential Breast Expression

    Science.gov (United States)

    Garbin, Catherine P.; Hartmann, Peter E.; Kent, Jacqueline C.

    2012-01-01

    Abstract Introduction Simultaneous (SIM) breast expression saves mothers time compared with sequential (SEQ) expression, but it remains unclear whether the two methods differ in milk output efficiency and efficacy. Subjects and Methods The Showmilk device (Medela AG, Baar, Switzerland) was used to measure milk output and milk ejection during breast expression (electric pump) in 31 Australian breastfeeding mothers of term infants (median age, 19 weeks [interquartile range, 10–33 weeks]). The order of expression type (SIM/SEQ) and breast (left/right) was randomized. Results SIM expression yielded more milk ejections (p≤0.001) and greater amounts of milk at 2, 5, and 10 minutes (p≤0.01) and removed a greater total amount of milk (p≤0.01) and percentage of available milk (p<0.05) than SEQ expression. After SIM expression the cream content of both the overall (8.3% [p≤0.05]) and postexpression (12.6% [p≤0.001]) milk were greater. During SEQ expression, the breast expressed first had a shorter time to 50% and 80% of the total amount of milk than the breast expressed second (p≤0.05), but, overall, a similar percentage of available milk was removed from both breasts. Conclusions SIM expression stimulated more milk ejections and was a more efficient and efficacious method of expression, yielding milk with a higher energy content. PMID:23039397

  15. The Relations of Mothers' Negative Expressivity to Children's Experience and Expression of Negative Emotion

    Science.gov (United States)

    Valiente, Carlos; Eisenberg, Nancy; Shepard, Stephanie A.; Fabes, Richard A.; Cumberland, Amanda J.; Losoya, Sandra H.; Spinrad, Tracy L.

    2004-01-01

    Guided by the heuristic model proposed by Eisenberg et al. [Psychol. Inq. 9 (1998) 241], we examined the relations of mothers' reported and observed negative expressivity to children's (N = 159; 74 girls; M age = 7.67 years) experience and expression of emotion. Children's experience and/or expression of emotion in response to a distressing film…

  16. Recognition, Expression, and Understanding Facial Expressions of Emotion in Adolescents with Nonverbal and General Learning Disabilities

    Science.gov (United States)

    Bloom, Elana; Heath, Nancy

    2010-01-01

    Children with nonverbal learning disabilities (NVLD) have been found to be worse at recognizing facial expressions than children with verbal learning disabilities (LD) and without LD. However, little research has been done with adolescents. In addition, expressing and understanding facial expressions is yet to be studied among adolescents with LD…

  17. ["Les Impatients": expression through art].

    Science.gov (United States)

    Lamontagne, Céline; Palardy, Lorraine

    2015-01-01

    The organization called "Les Impatients" was founded in 1992. Using a unique model, Les Impatients welcomes those with mental health issues who would like to express themselves through art. Les Impatients offers free creative workshops and encourages exchanges with the community through the sharing of its participants' creations. The name Les Impatients reinforces the idea that the organization does not consider those attending its workshops as patients, but rather creators who are eager to heal, develop their craft and find their place in society. The participants contribute to the collective objective of breaking down the stigma that surrounds mental illness.Les Impatients collaborates with various mental health organizations in Quebec, such as the Institut universitaire en santé mentale de Montréal (IUSMM) affiliated to the Université de Montréal, Douglas Mental Health University Institute (DMHUI), the Centre de santé et services sociaux Drummond (CSSS Drummond) and the Centre de santé et services sociaux Pierre-Boucher (CSSS Pierre-Boucher). Les Impatients offers more than 48 workshops in eight different locations to around 450 participants each week.Dissemination activities, remarkable events, original projects: Les Impatients stands out through its realizations. Examples are exhibitions, collections of love letters, comic books, CD, concerts, and reading nights. The organization's originality resides in the exploration of the links between the work of the participants and that of professional artists. An illustration of this interest is the annual Parle-moi d'amour auction-exhibition, which has been one of Les Impatients' major events since 1999.As part of its mission, Les Impatients conserves the works of art created by the participants during the workshops. Its collection includes more than 15,000 works of art from Les Impatients as well as pieces donated by collectors of unconventional art, commonly known as "art brut" or "outsider art". The

  18. Evaluation of an mRNA lipofection procedure for human dendritic cells and induction of cytotoxic T lymphocytes against enhanced green fluorescence protein.

    Science.gov (United States)

    Okano, Kozue; Fukui, Mikiko; Suehiro, Yutaka; Hamanaka, Yuichiro; Imai, Kohzoh; Hinoda, Yuji

    2003-01-01

    We utilized an mRNA lipofection procedure in human dendritic cells (DCs) and attempted to induce cytotoxic T lymphocytes (CTLs) against enhanced green fluorescence protein (EGFP). EGFP mRNA was transfected into phytohemagglutinin (PHA)-stimulated lymphocytes or adherent peripheral blood mononuclear cell-derived DCs using a liposomal reagent. Lipofection efficiency was measured by flow cytometry. In PHA-stimulated lymphocytes, increasing concentrations of liposome or mRNA increased EGFP expression levels by up to 64.4%, but caused a decrease in cell viability. A similar trend was also observed in DCs. For 70% DC viability, the concentration of liposomes was 24 microl/ml, and the mRNA concentration was 6 microg/ml. Under these conditions, ELISPOT and (51)Cr release assays were performed on CD8+ T cells stimulated twice with EGFP mRNA-transfected DCs. The number of interferon-gamma-producing cells was increased when the CD8+ T cells were cocultured for 24 h with PHA-stimulated lymphocytes transfected with EGFP mRNA. The level of specific lysis of EGFP mRNA-transfected DCs also increased to approximately 80%, with an effector to target ratio of 40:1. These data suggest that EGFP is immunogenic for human T cells, confirming that our lipofection procedure may be of use for inducing specific CTLs. Copyright 2003 S. Karger AG, Basel

  19. Micro-Expression Recognition Using Color Spaces.

    Science.gov (United States)

    Wang, Su-Jing; Yan, Wen-Jing; Li, Xiaobai; Zhao, Guoying; Zhou, Chun-Guang; Fu, Xiaolan; Yang, Minghao; Tao, Jianhua

    2015-12-01

    Micro-expressions are brief involuntary facial expressions that reveal genuine emotions and, thus, help detect lies. Because of their many promising applications, they have attracted the attention of researchers from various fields. Recent research reveals that two perceptual color spaces (CIELab and CIELuv) provide useful information for expression recognition. This paper is an extended version of our International Conference on Pattern Recognition paper, in which we propose a novel color space model, tensor independent color space (TICS), to help recognize micro-expressions. In this paper, we further show that CIELab and CIELuv are also helpful in recognizing micro-expressions, and we indicate why these three color spaces achieve better performance. A micro-expression color video clip is treated as a fourth-order tensor, i.e., a four-dimension array. The first two dimensions are the spatial information, the third is the temporal information, and the fourth is the color information. We transform the fourth dimension from RGB into TICS, in which the color components are as independent as possible. The combination of dynamic texture and independent color components achieves a higher accuracy than does that of RGB. In addition, we define a set of regions of interests (ROIs) based on the facial action coding system and calculated the dynamic texture histograms for each ROI. Experiments are conducted on two micro-expression databases, CASME and CASME 2, and the results show that the performances for TICS, CIELab, and CIELuv are better than those for RGB or gray.

  20. Glucose transporters: expression, regulation and cancer

    Directory of Open Access Journals (Sweden)

    RODOLFO A. MEDINA

    2002-01-01

    Full Text Available Mammalian cells depend on glucose as a major substrate for energy production. Glucose is transported into the cell via facilitative glucose transporters (GLUT present in all cell types. Many GLUT isoforms have been described and their expression is cell-specific and subject to hormonal and environmental control. The kinetic properties and substrate specificities of the different isoforms are specifically suited to the energy requirements of the particular cell types. Due to the ubiquitousness of these transporters, their differential expression is involved in various disease states such as diabetes, ischemia and cancer. The majority of cancers and isolated cancer cell lines over-express the GLUT family members which are present in the respective tissue of origin under non-cancerous conditions. Moreover, due to the requirement of energy to feed uncontrolled proliferation, cancer cells often express GLUTs which under normal conditions would not be present in these tissues. This over-expression is predominantly associated with the likelihood of metastasis and hence poor patient prognosis. This article presents a review of the current literature on the regulation and expression of GLUT family members and has compiled clinical and research data on GLUT expression in human cancers and in isolated human cancer cell lines.

  1. Regular Expression Matching and Operational Semantics

    Directory of Open Access Journals (Sweden)

    Asiri Rathnayake

    2011-08-01

    Full Text Available Many programming languages and tools, ranging from grep to the Java String library, contain regular expression matchers. Rather than first translating a regular expression into a deterministic finite automaton, such implementations typically match the regular expression on the fly. Thus they can be seen as virtual machines interpreting the regular expression much as if it were a program with some non-deterministic constructs such as the Kleene star. We formalize this implementation technique for regular expression matching using operational semantics. Specifically, we derive a series of abstract machines, moving from the abstract definition of matching to increasingly realistic machines. First a continuation is added to the operational semantics to describe what remains to be matched after the current expression. Next, we represent the expression as a data structure using pointers, which enables redundant searches to be eliminated via testing for pointer equality. From there, we arrive both at Thompson's lockstep construction and a machine that performs some operations in parallel, suitable for implementation on a large number of cores, such as a GPU. We formalize the parallel machine using process algebra and report some preliminary experiments with an implementation on a graphics processor using CUDA.

  2. The Communicative Function of Sad Facial Expressions.

    Science.gov (United States)

    Reed, Lawrence Ian; DeScioli, Peter

    2017-01-01

    What are the communicative functions of sad facial expressions? Research shows that people feel sadness in response to losses but it's unclear whether sad expressions function to communicate losses to others and if so, what makes these signals credible. Here we use economic games to test the hypothesis that sad expressions lend credibility to claims of loss. Participants play the role of either a proposer or recipient in a game with a fictional backstory and real monetary payoffs. The proposers view a (fictional) video of the recipient's character displaying either a neutral or sad expression paired with a claim of loss. The proposer then decided how much money to give to the recipient. In three experiments, we test alternative theories by using situations in which the recipient's losses were uncertain (Experiment 1), the recipient's losses were certain (Experiment 2), or the recipient claims failed gains rather than losses (Experiment 3). Overall, we find that participants gave more money to recipients who displayed sad expressions compared to neutral expressions, but only under conditions of uncertain loss. This finding supports the hypothesis that sad expressions function to increase the credibility of claims of loss.

  3. Expressive Dissonance: When Emotional Inconsistency Arouses Dissonance

    Directory of Open Access Journals (Sweden)

    Audrey Pelt

    2018-05-01

    Full Text Available The aim of the two studies was to explore a new dissonance paradigm–expressive dissonance–based on the inconsistency between what people feel and what people express behaviorally. Expressive dissonance was aroused by asking participants to watch a film with a high emotional content, either positive (joy or negative (sadness. In the no-dissonance condition, they received the instruction to naturally watch the film. In the expressive dissonance condition, they received the instruction to facially express emotions that were the opposite of what they felt. We expected that the expressive dissonance situation would: 1 require cognitive resources leading to a decrease in cognitive performance (studies 1 and 2; 2 be accompanied by emotional regulation strategies (study 1; 3 be accompanied by an increase in dissonance-related affects (study 2. Although our results (studies 1 and 2 corroborated those obtained previously in terms of performance, they also showed that participants in the expressive dissonance situation use emotional regulation strategies: exaggeration and suppression (study 1, and that they felt self-directed negative affects (study 2, just like the participants in a cognitive dissonance situation. These first results allowed us to establish a theoretical bridge between the theories of emotions–particularly those related to the emotional regulation processes–and to widen the scope of relevance of the dissonance theory.

  4. Gene expression and gene therapy imaging

    International Nuclear Information System (INIS)

    Rome, Claire; Couillaud, Franck; Moonen, Chrit T.W.

    2007-01-01

    The fast growing field of molecular imaging has achieved major advances in imaging gene expression, an important element of gene therapy. Gene expression imaging is based on specific probes or contrast agents that allow either direct or indirect spatio-temporal evaluation of gene expression. Direct evaluation is possible with, for example, contrast agents that bind directly to a specific target (e.g., receptor). Indirect evaluation may be achieved by using specific substrate probes for a target enzyme. The use of marker genes, also called reporter genes, is an essential element of MI approaches for gene expression in gene therapy. The marker gene may not have a therapeutic role itself, but by coupling the marker gene to a therapeutic gene, expression of the marker gene reports on the expression of the therapeutic gene. Nuclear medicine and optical approaches are highly sensitive (detection of probes in the picomolar range), whereas MRI and ultrasound imaging are less sensitive and require amplification techniques and/or accumulation of contrast agents in enlarged contrast particles. Recently developed MI techniques are particularly relevant for gene therapy. Amongst these are the possibility to track gene therapy vectors such as stem cells, and the techniques that allow spatiotemporal control of gene expression by non-invasive heating (with MRI guided focused ultrasound) and the use of temperature sensitive promoters. (orig.)

  5. Expression of multidrug resistance proteins in retinoblastoma

    Directory of Open Access Journals (Sweden)

    Swati Shukla

    2017-11-01

    Full Text Available AIM: To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS: Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1 expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION: Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

  6. Facial Expression Recognition Through Machine Learning

    Directory of Open Access Journals (Sweden)

    Nazia Perveen

    2015-08-01

    Full Text Available Facial expressions communicate non-verbal cues which play an important role in interpersonal relations. Automatic recognition of facial expressions can be an important element of normal human-machine interfaces it might likewise be utilized as a part of behavioral science and in clinical practice. In spite of the fact that people perceive facial expressions for all intents and purposes immediately solid expression recognition by machine is still a challenge. From the point of view of automatic recognition a facial expression can be considered to comprise of disfigurements of the facial parts and their spatial relations or changes in the faces pigmentation. Research into automatic recognition of the facial expressions addresses the issues encompassing the representation and arrangement of static or dynamic qualities of these distortions or face pigmentation. We get results by utilizing the CVIPtools. We have taken train data set of six facial expressions of three persons and for train data set purpose we have total border mask sample 90 and 30 border mask sample for test data set purpose and we use RST- Invariant features and texture features for feature analysis and then classified them by using k- Nearest Neighbor classification algorithm. The maximum accuracy is 90.

  7. Family emotional expressiveness and family structure

    Directory of Open Access Journals (Sweden)

    Čotar-Konrad Sonja

    2016-01-01

    Full Text Available The present paper scrutinizes the relationship between family emotional expressiveness (i.e., the tendency to express dominant and/or submissive positive and negative emotions and components of family structure as proposed in Olson’s Circumplex model (i.e., cohesion and flexibility, family communication, and satisfaction in families with adolescents. The study was conducted on a sample of 514 Slovenian adolescents, who filled out two questionnaires: the Slovenian version of Family Emotional Expressiveness - FEQ and FACES IV. The results revealed that all four basic dimensions of family functioning were significantly associated with higher/more frequent expressions of positive submissive emotions, as well as with lower/less frequent expressions of negative dominant emotions. Moreover, expressions of negative submissive emotions explained a small, but significant amount of variance in three out of four family functioning variables (satisfaction, flexibility, and communication. The importance of particular aspects of emotional expressiveness for family cohesion, flexibility, communication, and satisfaction is discussed, and the relevance of present findings for family counselling is outlined.

  8. The Communicative Function of Sad Facial Expressions

    Directory of Open Access Journals (Sweden)

    Lawrence Ian Reed

    2017-03-01

    Full Text Available What are the communicative functions of sad facial expressions? Research shows that people feel sadness in response to losses but it’s unclear whether sad expressions function to communicate losses to others and if so, what makes these signals credible. Here we use economic games to test the hypothesis that sad expressions lend credibility to claims of loss. Participants play the role of either a proposer or recipient in a game with a fictional backstory and real monetary payoffs. The proposers view a (fictional video of the recipient’s character displaying either a neutral or sad expression paired with a claim of loss. The proposer then decided how much money to give to the recipient. In three experiments, we test alternative theories by using situations in which the recipient’s losses were uncertain (Experiment 1, the recipient’s losses were certain (Experiment 2, or the recipient claims failed gains rather than losses (Experiment 3. Overall, we find that participants gave more money to recipients who displayed sad expressions compared to neutral expressions, but only under conditions of uncertain loss. This finding supports the hypothesis that sad expressions function to increase the credibility of claims of loss.

  9. Expression of multidrug resistance proteins in retinoblastoma.

    Science.gov (United States)

    Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

    2017-01-01

    To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

  10. Expression of multidrug resistance proteins in retinoblastoma

    Science.gov (United States)

    Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

    2017-01-01

    AIM To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy. PMID:29181307

  11. A comparative gene expression database for invertebrates

    Directory of Open Access Journals (Sweden)

    Ormestad Mattias

    2011-08-01

    Full Text Available Abstract Background As whole genome and transcriptome sequencing gets cheaper and faster, a great number of 'exotic' animal models are emerging, rapidly adding valuable data to the ever-expanding Evo-Devo field. All these new organisms serve as a fantastic resource for the research community, but the sheer amount of data, some published, some not, makes detailed comparison of gene expression patterns very difficult to summarize - a problem sometimes even noticeable within a single lab. The need to merge existing data with new information in an organized manner that is publicly available to the research community is now more necessary than ever. Description In order to offer a homogenous way of storing and handling gene expression patterns from a variety of organisms, we have developed the first web-based comparative gene expression database for invertebrates that allows species-specific as well as cross-species gene expression comparisons. The database can be queried by gene name, developmental stage and/or expression domains. Conclusions This database provides a unique tool for the Evo-Devo research community that allows the retrieval, analysis and comparison of gene expression patterns within or among species. In addition, this database enables a quick identification of putative syn-expression groups that can be used to initiate, among other things, gene regulatory network (GRN projects.

  12. Adaptive Evolution of Gene Expression in Drosophila.

    Science.gov (United States)

    Nourmohammad, Armita; Rambeau, Joachim; Held, Torsten; Kovacova, Viera; Berg, Johannes; Lässig, Michael

    2017-08-08

    Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. Here, we present evidence that adaptation dominates the evolution of gene expression levels in flies. We show that 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. Our results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, we identify a large group of genes with sex-specific adaptation of expression, which predominantly occurs in males. Our analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Cyclin D1 expression in prostate carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, R.A.; Ravinal, R.C.; Costa, R.S.; Lima, M.S. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Patologia, Ribeirão Preto, SP, Brasil, Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Tucci, S. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Cirurgia e Anatomia, Divisão de Urologia, Ribeirão Preto, SP, Brasil, Divisão de Urologia, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Muglia, V.F. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Medicina Interna (Centro de Ciência da Imagem), Ribeirão Preto, SP, Brasil, Departamento de Medicina Interna (Centro de Ciência da Imagem), Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Reis, R.B. Dos [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Cirurgia e Anatomia, Divisão de Urologia, Ribeirão Preto, SP, Brasil, Divisão de Urologia, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Silva, G.E.B. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Patologia, Ribeirão Preto, SP, Brasil, Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2014-05-09

    The purpose of this study was to investigate the relationship between cyclin D1 expression and clinicopathological parameters in patients with prostate carcinoma. We assessed cyclin D1 expression by conventional immunohistochemistry in 85 patients who underwent radical prostatectomy for prostate carcinoma and 10 normal prostate tissue samples retrieved from autopsies. We measured nuclear immunostaining in the entire tumor area and based the results on the percentage of positive tumor cells. The preoperative prostate-specific antigen (PSA) level was 8.68±5.16 ng/mL (mean±SD). Cyclin D1 staining was positive (cyclin D1 expression in >5% of tumor cells) in 64 cases (75.4%) and negative (cyclin D1 expression in ≤5% of tumor cells) in 21 cases (including 15 cases with no immunostaining). Normal prostate tissues were negative for cyclin D1. Among patients with a high-grade Gleason score (≥7), 86% of patients demonstrated cyclin D1 immunostaining of >5% (P<0.05). In the crude analysis of cyclin D1 expression, the high-grade Gleason score group showed a mean expression of 39.6%, compared to 26.9% in the low-grade Gleason score group (P<0.05). Perineural invasion tended to be associated with cyclin D1 expression (P=0.07), whereas cyclin D1 expression was not associated with PSA levels or other parameters. Our results suggest that high cyclin D1 expression could be a potential marker for tumor aggressiveness.

  14. Cyclin D1 expression in prostate carcinoma

    International Nuclear Information System (INIS)

    Pereira, R.A.; Ravinal, R.C.; Costa, R.S.; Lima, M.S.; Tucci, S.; Muglia, V.F.; Reis, R.B. Dos; Silva, G.E.B.

    2014-01-01

    The purpose of this study was to investigate the relationship between cyclin D1 expression and clinicopathological parameters in patients with prostate carcinoma. We assessed cyclin D1 expression by conventional immunohistochemistry in 85 patients who underwent radical prostatectomy for prostate carcinoma and 10 normal prostate tissue samples retrieved from autopsies. We measured nuclear immunostaining in the entire tumor area and based the results on the percentage of positive tumor cells. The preoperative prostate-specific antigen (PSA) level was 8.68±5.16 ng/mL (mean±SD). Cyclin D1 staining was positive (cyclin D1 expression in >5% of tumor cells) in 64 cases (75.4%) and negative (cyclin D1 expression in ≤5% of tumor cells) in 21 cases (including 15 cases with no immunostaining). Normal prostate tissues were negative for cyclin D1. Among patients with a high-grade Gleason score (≥7), 86% of patients demonstrated cyclin D1 immunostaining of >5% (P<0.05). In the crude analysis of cyclin D1 expression, the high-grade Gleason score group showed a mean expression of 39.6%, compared to 26.9% in the low-grade Gleason score group (P<0.05). Perineural invasion tended to be associated with cyclin D1 expression (P=0.07), whereas cyclin D1 expression was not associated with PSA levels or other parameters. Our results suggest that high cyclin D1 expression could be a potential marker for tumor aggressiveness

  15. Effect of lentiviruses carrying enhanced green fluorescent protein injected into the scala media through a cochleostomy in rats.

    Science.gov (United States)

    Wei, Yan; Fu, Yong; Liu, Shaosheng; Xia, Guihua; Pan, Song

    2013-01-01

    The purposes of the current study were to assess the feasibility of post-auricular microinjection of lentiviruses carrying enhanced green fluorescent protein (EGFP) into the scala media through cochleostomies in rats, determine the expression of viral gene in the cochlea, and record the post-operative changes in the number and auditory function of cochlear hair cells (HCs). Healthy rats were randomly divided into two groups. The left ears of the animals in group I were injected with lentivirus carrying EGFP (n=10) via scala media lateral wall cochleostomies, and the left ears of the animals in group II were similarly injected with artificial endolymph (n=10). Prior to and 30 days post-injection, auditory function was assessed with click-auditory brainstem response (ABR) testing, EGFP expression was determined with cochlear frozen sections under fluorescence microscopy, and survival of HCs was estimated based on whole mount preparations. Thirty days after surgery, click-ABR testing revealed that there were significant differences in the auditory function, EGFP expression, and survival of HCs in the left ears before and after surgery in the same rats from each group. In group I, EGFP was noted in the strial marginal cells of the scala media, the organ of Corti, spiral nerves, and spiral ganglion cells. Lentiviruses were successfully introduced into the scala media through cochleostomies in rats, and the EGFP reporter gene was efficiently expressed in the organ of Corti, spiral nerves, and spiral ganglion cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Effects of Expressive Writing on Psychological and Physical Health: The Moderating Role of Emotional Expressivity

    Science.gov (United States)

    Haltom, Kate E.; Mulvenna, Catherine M.; Lieberman, Matthew D.; Stanton, Annette L.

    2013-01-01

    The current study assessed main effects and moderators (including emotional expressiveness, emotional processing and ambivalence over emotional expression) of the effects of expressive writing in a sample of healthy adults. Young adult participants (N = 116) were randomly assigned to write for 20 minutes on four occasions about deepest thoughts and feelings regarding their most stressful/traumatic event in the past five years (expressive writing) or about a control topic (control). Dependent variables were indicators of anxiety, depression, and physical symptoms. No significant effects of writing condition were evident on anxiety, depressive symptoms, or physical symptoms. Emotional expressiveness emerged as a significant moderator of anxiety outcomes, however. Within the expressive writing group, participants high in expressiveness evidenced a significant reduction in anxiety at three-month follow-up, and participants low in expressiveness showed a significant increase in anxiety. Expressiveness did not predict change in anxiety in the control group. These findings on anxiety are consistent with the matching hypothesis, which suggests that matching a person’s naturally elected coping approach with an assigned intervention is beneficial. These findings also suggest that expressive writing about a stressful event may be contraindicated for individuals who do not typically express emotions. PMID:23742666

  17. Expression-dependent susceptibility to face distortions in processing of facial expressions of emotion.

    Science.gov (United States)

    Guo, Kun; Soornack, Yoshi; Settle, Rebecca

    2018-03-05

    Our capability of recognizing facial expressions of emotion under different viewing conditions implies the existence of an invariant expression representation. As natural visual signals are often distorted and our perceptual strategy changes with external noise level, it is essential to understand how expression perception is susceptible to face distortion and whether the same facial cues are used to process high- and low-quality face images. We systematically manipulated face image resolution (experiment 1) and blur (experiment 2), and measured participants' expression categorization accuracy, perceived expression intensity and associated gaze patterns. Our analysis revealed a reasonable tolerance to face distortion in expression perception. Reducing image resolution up to 48 × 64 pixels or increasing image blur up to 15 cycles/image had little impact on expression assessment and associated gaze behaviour. Further distortion led to decreased expression categorization accuracy and intensity rating, increased reaction time and fixation duration, and stronger central fixation bias which was not driven by distortion-induced changes in local image saliency. Interestingly, the observed distortion effects were expression-dependent with less deterioration impact on happy and surprise expressions, suggesting this distortion-invariant facial expression perception might be achieved through the categorical model involving a non-linear configural combination of local facial features. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Regulation of Gene Expression in Protozoa Parasites

    Directory of Open Access Journals (Sweden)

    Consuelo Gomez

    2010-01-01

    Full Text Available Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.

  19. Regulation of gene expression in protozoa parasites.

    Science.gov (United States)

    Gomez, Consuelo; Esther Ramirez, M; Calixto-Galvez, Mercedes; Medel, Olivia; Rodríguez, Mario A

    2010-01-01

    Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.

  20. Lunar Plants Prototype for Moon Express

    Data.gov (United States)

    National Aeronautics and Space Administration — The goal of our project is to bring the first full life cycle to the moon: to demonstrate germination of plants in lunar gravity and radiation.The Moon Express...

  1. Proteomic Analysis of Bacterial Expression Profiles Following ...

    African Journals Online (AJOL)

    mass spectrometry (GC-MS) were performed to determine the phytochemicals in the active fraction. Results: Five differentially expressed bacterial proteins (four from Escherichia coli and one from Staphylococcus aureus), were identified via ...

  2. Inferring gene networks from discrete expression data

    KAUST Repository

    Zhang, L.; Mallick, B. K.

    2013-01-01

    graphical models applied to continuous data, which give a closedformmarginal likelihood. In this paper,we extend network modeling to discrete data, specifically data from serial analysis of gene expression, and RNA-sequencing experiments, both of which

  3. Gene Expression and Microarray Investigation of Dendrobium ...

    African Journals Online (AJOL)

    blood glucose > 16.7 mmol/L were used as the model group and treated with Dendrobium mixture. (DEN ... Keywords: Diabetes, Gene expression, Dendrobium mixture, Microarray testing ..... homeostasis in airway smooth muscle. Am J.

  4. Decreased decorin expression in the tumor microenvironment

    International Nuclear Information System (INIS)

    Bozoky, Benedek; Savchenko, Andrii; Guven, Hayrettin; Ponten, Fredrik; Klein, George; Szekely, Laszlo

    2014-01-01

    Decorin is a small leucine-rich proteoglycan, synthesized and deposited by fibroblasts in the stroma where it binds to collagen I. It sequesters several growth factors and antagonizes numerous members of the receptor tyrosine kinase family. In experimental murine systems, it acted as a potent tumor suppressor. Examining the Human Protein Atlas online database of immunostained tissue samples we have surveyed decorin expression in silico in several different tumor types, comparing them with corresponding normal tissues. We found that decorin is abundantly secreted and deposited in normal connective tissue but its expression is consistently decreased in the tumor microenvironment. We developed a software to quantitate the difference in expression. The presence of two closely related proteoglycans in the newly formed tumor stroma indicated that the decreased decorin expression was not caused by the delay in proteoglycan deposition in the newly formed connective tissue surrounding the tumor

  5. SOX4 expression in bladder carcinoma

    DEFF Research Database (Denmark)

    Aaboe, Mads; Birkenkamp-Demtroder, Karin; Wiuf, Carsten

    2006-01-01

    The human transcription factor SOX4 was 5-fold up-regulated in bladder tumors compared with normal tissue based on whole-genome expression profiling of 166 clinical bladder tumor samples and 27 normal urothelium samples. Using a SOX4-specific antibody, we found that the cancer cells expressed...... in the clinical bladder material and a small subset of the genes showed a high correlation to SOX4 expression. The present data suggest a role of SOX4 in the bladder cancer disease....... the SOX4 protein and, thus, did an evaluation of SOX4 protein expression in 2,360 bladder tumors using a tissue microarray with clinical annotation. We found a correlation (P bladder cell line HU609, SOX4...

  6. Identification of genes showing differential expression profile ...

    Indian Academy of Sciences (India)

    3Department of Natural Sciences, International Christian University, Mitaka, Tokyo 181-8585, Japan ... the changes of expression predicted from gene function suggested association ... ate School of Science and Technology, Niigata University.

  7. Drosophila melanogaster gene expression changes after spaceflight.

    Data.gov (United States)

    National Aeronautics and Space Administration — Gene expression levels were determined in 3rd instar and adult Drosophila melanogaster reared during spaceflight to elucidate the genetic and molecular mechanisms...

  8. Expression of maize prolamins in Escherichia Coli

    International Nuclear Information System (INIS)

    Wang, Szu-zhen; Esen, Asim

    1985-01-01

    We have constructed a cDNA expression library of developing corn (Zea manys L.) endosperm using plasmid pUC8 as vector and Escherichia coli strain DH1 as host. The expression library was screened with non-radioactive immunological probes to detect the expression of gamma-zein and alpha-zein. When anti-gamma-zein antibody was used as the probe, 23 colonies gave positive reactions. The lengths of cDNA inserts of the 23 colonies were found to be 250-900 base pairs. When anti-alpha zein antibody was used, however, fewer colonies gave positive reactions. The library was also screened by colony-hybridization with 32 P-labeled DNA probes. Based on immunological and hybridization screening of the library and other evidence, we conclude that alpha-zein was either toxic to E. coli cells or rapidly degraded whereas gamma-zein and its fragments were readily expressed. (author)

  9. Determinants of human adipose tissue gene expression

    DEFF Research Database (Denmark)

    Viguerie, Nathalie; Montastier, Emilie; Maoret, Jean-José

    2012-01-01

    weight maintenance diets. For 175 genes, opposite regulation was observed during calorie restriction and weight maintenance phases, independently of variations in body weight. Metabolism and immunity genes showed inverse profiles. During the dietary intervention, network-based analyses revealed strong...... interconnection between expression of genes involved in de novo lipogenesis and components of the metabolic syndrome. Sex had a marked influence on AT expression of 88 transcripts, which persisted during the entire dietary intervention and after control for fat mass. In women, the influence of body mass index...... on expression of a subset of genes persisted during the dietary intervention. Twenty-two genes revealed a metabolic syndrome signature common to men and women. Genetic control of AT gene expression by cis signals was observed for 46 genes. Dietary intervention, sex, and cis genetic variants independently...

  10. Exertional Heat Illness and Human Gene Expression

    National Research Council Canada - National Science Library

    Sonna, L.A; Sawka, M. N; Lilly, C. M

    2007-01-01

    Microarray analysis of gene expression at the level of RNA has generated new insights into the relationship between cellular responses to acute heat shock in vitro, exercise, and exertional heat illness...

  11. Deriving Trading Rules Using Gene Expression Programming

    Directory of Open Access Journals (Sweden)

    Adrian VISOIU

    2011-01-01

    Full Text Available This paper presents how buy and sell trading rules are generated using gene expression programming with special setup. Market concepts are presented and market analysis is discussed with emphasis on technical analysis and quantitative methods. The use of genetic algorithms in deriving trading rules is presented. Gene expression programming is applied in a form where multiple types of operators and operands are used. This gives birth to multiple gene contexts and references between genes in order to keep the linear structure of the gene expression programming chromosome. The setup of multiple gene contexts is presented. The case study shows how to use the proposed gene setup to derive trading rules encoded by Boolean expressions, using a dataset with the reference exchange rates between the Euro and the Romanian leu. The conclusions highlight the positive results obtained in deriving useful trading rules.

  12. The human endolymphatic sac expresses natriuretic peptides

    DEFF Research Database (Denmark)

    Møller, Martin Nue; Kirkeby, Svend; Vikeså, Jonas

    2017-01-01

    : Several natriuretic peptides were found expressed significantly in the ES, including uroguanylin and brain natriuretic peptide, but also peptides regulating vascular tone, including adrenomedullin 2. In addition, both neurophysin and oxytocin (OXT) were found significantly expressed. All peptides were...... verified by immunohistochemistry. CONCLUSION: The present data support the hypothesis that the human ES may have an endocrine/paracrine capacity through expression of several peptides with potent natriuretic activity. Furthermore, the ES may influence the hypothalamo-pituitary-adrenal axis and may regulate...... vasopressin receptors and aquaporin-2 channels in the inner ear via OXT expression. We hypothesize that the ES is likely to regulate inner ear endolymphatic homeostasis, possibly through secretion of several peptides, but it may also influence systemic and/or intracranial blood pressure through direct...

  13. Expression Profiling of Tyrosine Kinase Genes

    National Research Council Canada - National Science Library

    Weier, Heinz

    2000-01-01

    ... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

  14. Regulation of meiotic gene expression in plants

    Directory of Open Access Journals (Sweden)

    Adele eZhou

    2014-08-01

    Full Text Available With the recent advances in genomics and sequencing technologies, databases of transcriptomes representing many cellular processes have been built. Meiotic transcriptomes in plants have been studied in Arabidopsis thaliana, rice (Oryza sativa, wheat (Triticum aestivum, petunia (Petunia hybrida, sunflower (Helianthus annuus, and maize (Zea mays. Studies in all organisms, but particularly in plants, indicate that a very large number of genes are expressed during meiosis, though relatively few of them seem to be required for the completion of meiosis. In this review, we focus on gene expression at the RNA level and analyze the meiotic transcriptome datasets and explore expression patterns of known meiotic genes to elucidate how gene expression could be regulated during meiosis. We also discuss mechanisms, such as chromatin organization and non-coding RNAs, that might be involved in the regulation of meiotic transcription patterns.

  15. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    Supplementary figure 1. Protein expression dynamics observed in Experiment, Synchronous and. Asynchronous simulation. .... molecular basis for T cell suppression by IL-10: CD28-asso- ciated IL-10 receptor inhibits CD28 tyrosine ...

  16. Identification of genes preferentially expressed during

    African Journals Online (AJOL)

    雨林木风

    2012-08-16

    Aug 16, 2012 ... The suppression subtractive hybridization (SSH) method conducted to generate ... which showed the lack of genomic information currently available for lily. ..... characterization of genes expressed during somatic embryo.

  17. Subcellular localization of Bombyx mori ribosomal protein S3a and ...

    African Journals Online (AJOL)

    USER

    2010-04-05

    Apr 5, 2010 ... In the present study, using a BV/PH-Bms3a-EGFP, we found that Bombyx mori ribosomal protein S3a. (BmS3a) with EGFP fused to its C-terminal, was predominantly localized in the cytoplasm of B. mori cells. Subsequently, to investigate the effect of BmS3a over-expression on BmNPV infection both at the.

  18. An atlas of Caenorhabditis elegans chemoreceptor expression.

    Directory of Open Access Journals (Sweden)

    Berta Vidal

    2018-01-01

    Full Text Available One goal of modern day neuroscience is the establishment of molecular maps that assign unique features to individual neuron types. Such maps provide important starting points for neuron classification, for functional analysis, and for developmental studies aimed at defining the molecular mechanisms of neuron identity acquisition and neuron identity diversification. In this resource paper, we describe a nervous system-wide map of the potential expression sites of 244 members of the largest gene family in the C. elegans genome, rhodopsin-like (class A G-protein-coupled receptor (GPCR chemoreceptors, using classic gfp reporter gene technology. We cover representatives of all sequence families of chemoreceptor GPCRs, some of which were previously entirely uncharacterized. Most reporters are expressed in a very restricted number of cells, often just in single cells. We assign GPCR reporter expression to all but two of the 37 sensory neuron classes of the sex-shared, core nervous system. Some sensory neurons express a very small number of receptors, while others, particularly nociceptive neurons, coexpress several dozen GPCR reporter genes. GPCR reporters are also expressed in a wide range of inter- and motorneurons, as well as non-neuronal cells, suggesting that GPCRs may constitute receptors not just for environmental signals, but also for internal cues. We observe only one notable, frequent association of coexpression patterns, namely in one nociceptive amphid (ASH and two nociceptive phasmid sensory neurons (PHA, PHB. We identified GPCRs with sexually dimorphic expression and several GPCR reporters that are expressed in a left/right asymmetric manner. We identified a substantial degree of GPCR expression plasticity; particularly in the context of the environmentally-induced dauer diapause stage when one third of all tested GPCRs alter the cellular specificity of their expression within and outside the nervous system. Intriguingly, in a number of

  19. Fast and compact regular expression matching

    DEFF Research Database (Denmark)

    Bille, Philip; Farach-Colton, Martin

    2008-01-01

    We study 4 problems in string matching, namely, regular expression matching, approximate regular expression matching, string edit distance, and subsequence indexing, on a standard word RAM model of computation that allows logarithmic-sized words to be manipulated in constant time. We show how...... to improve the space and/or remove a dependency on the alphabet size for each problem using either an improved tabulation technique of an existing algorithm or by combining known algorithms in a new way....

  20. Unsupervised learning of facial expression components

    OpenAIRE

    Egede, Joy Onyekachukwu

    2013-01-01

    The face is one of the most important means of non-verbal communication. A lot of information can be gotten about the emotional state of a person just by merely observing their facial expression. This is relatively easy in face to face communication but not so in human computer interaction. Supervised learning has been widely used by researchers to train machines to recognise facial expressions just like humans. However, supervised learning has significant limitations one of which is the fact...

  1. Gauge choice in Witten's energy expression

    International Nuclear Information System (INIS)

    Parker, T.H.

    1985-01-01

    Witten's equation Dpsi=0 can be interpreted as a gauge fixing condition for classical supergravity. We rigorously prove the existence of asymptotically constant solutions of the more general gauge condition Dpsi=Apsi for almost all endomorphisms A of the spin bundle. Each gives an expression for the gravitational energy similar to Witten's. These include the choice A=√R, which yields the particularly elegant energy expression first noticed by Deser. (orig.)

  2. Analytical expressions for the electron backscattering coefficient

    International Nuclear Information System (INIS)

    August, H.J.; Wernisch, J.

    1989-01-01

    Several analytical expressions for the electron backscattering coefficient for massive homogeneous samples are compared with experimental data, directing special attention to the dependence of this quantity on the electron acceleration energy. It is shown that this dependence generally cannot be neglected. The expression proposed by Hunger and Kuechler turns out to be better than that of Love and Scott, although even the better formula can be slightly improved by a small modification. (author)

  3. Le tappe italiane del Trimble Express 2010

    Directory of Open Access Journals (Sweden)

    Sandra Leonardi

    2010-03-01

    Full Text Available Trimble Express stopped over Italy Trimble Express stopped in Italy, in 6 cities. The european road show has been set up to promote the latest news regarding Trimble technologies such as Trimble Access, the software that offers to survey teams a new approach in surveying; Trimble Access expedites data collection, processing, analysis, and delivery through improved workflows, collaboration and control.

  4. Le tappe italiane del Trimble Express 2010

    Directory of Open Access Journals (Sweden)

    Sandra Leonardi

    2010-03-01

    Full Text Available Trimble Express stopped over ItalyTrimble Express stopped in Italy, in 6 cities. The european road show has been set up to promote the latest news regarding Trimble technologies such as Trimble Access, the software that offers to survey teams a new approach in surveying; Trimble Access expedites data collection, processing, analysis, and delivery through improved workflows, collaboration and control.

  5. Hepatocyte specific expression of human cloned genes

    Energy Technology Data Exchange (ETDEWEB)

    Cortese, R

    1986-01-01

    A large number of proteins are specifically synthesized in the hepatocyte. Only the adult liver expresses the complete repertoire of functions which are required at various stages during development. There is therefore a complex series of regulatory mechanisms responsible for the maintenance of the differentiated state and for the developmental and physiological variations in the pattern of gene expression. Human hepatoma cell lines HepG2 and Hep3B display a pattern of gene expression similar to adult and fetal liver, respectively; in contrast, cultured fibroblasts or HeLa cells do not express most of the liver specific genes. They have used these cell lines for transfection experiments with cloned human liver specific genes. DNA segments coding for alpha1-antitrypsin and retinol binding protein (two proteins synthesized both in fetal and adult liver) are expressed in the hepatoma cell lines HepG2 and Hep3B, but not in HeLa cells or fibroblasts. A DNA segment coding for haptoglobin (a protein synthesized only after birth) is only expressed in the hepatoma cell line HepG2 but not in Hep3B nor in non hepatic cell lines. The information for tissue specific expression is located in the 5' flanking region of all three genes. In vivo competition experiments show that these DNA segments bind to a common, apparently limiting, transacting factor. Conventional techniques (Bal deletions, site directed mutagenesis, etc.) have been used to precisely identify the DNA sequences responsible for these effects. The emerging picture is complex: they have identified multiple, separate transcriptional signals, essential for maximal promoter activation and tissue specific expression. Some of these signals show a negative effect on transcription in fibroblast cell lines.

  6. Colocalization of connexin 36 and corticotropin-releasing hormone in the mouse brain

    Directory of Open Access Journals (Sweden)

    Ribeiro Ana C

    2009-04-01

    Full Text Available Abstract Background Gap junction proteins, connexins, are expressed in most endocrine and exocrine glands in the body and are at least in some glands crucial for the hormonal secretion. To what extent connexins are expressed in neurons releasing hormones or neuropeptides from or within the central nervous system is, however, unknown. Previous studies provide indirect evidence for gap junction coupling between subsets of neuropeptide-containing neurons in the paraventricular nucleus (PVN of the hypothalamus. Here we employ double labeling and retrograde tracing methods to investigate to what extent neuroendocrine and neuropeptide-containing neurons of the hypothalamus and brainstem express the neuronal gap junction protein connexin 36. Results Western blot analysis showed that connexin 36 is expressed in the PVN. In bacterial artificial chromosome transgenic mice, which specifically express the reporter gene Enhanced Green Fluorescent Protein (EGFP under the control of the connexin 36 gene promoter, EGFP expression was detected in magnocellular (neuroendocrine and in parvocellular neurons of the PVN. Although no EGFP/connexin36 expression was seen in neurons containing oxytocin or vasopressin, EGFP/connexin36 was found in subsets of PVN neurons containing corticotropin-releasing hormone (CRH, and in somatostatin neurons located along the third ventricle. Moreover, CRH neurons in brainstem areas, including the lateral parabrachial nucleus, also expressed EGFP/connexin 36. Conclusion Our data indicate that connexin 36 is expressed in subsets of neuroendocrine and CRH neurons in specific nuclei of the hypothalamus and brainstem.

  7. Fluoxetin Upregulates Connexin 43 Expression in Astrocyte

    Directory of Open Access Journals (Sweden)

    Hossein Mostafavi

    2014-02-01

    Full Text Available Introduction: Recent studies have shown that astrocytes play major roles in normal and disease condition of the central nervous system including multiple sclerosis (MS. Molecular target therapy studies in MS have revealed that connexin-43 (Cx43 and Aquaporin-4 (AQP4 contents of astrocytes undergo expression alteration. Fluoxetine had some effects in MS patients unrelated to its known antidepressant effects. Some of fluoxetine effects were attributed to its capability of cAMP signaling pathway stimulation. This study aimed to investigate possible acute effects of fluoxetine on Cx43 and AQP4 expression in astrocyte.  Methods: Astrocytoma cells were treated for 24 hours with fluoxetine (10 and 20 &mug/ml with or without adenyl cyclase (AC and protein kinase A (PKA inhibition. Cx43 expression at both mRNA and protein levels and AQP4 expression at mRNA level were evaluated.  Results: Acquired results showed that fluoxetine with and without AC and PKA inhibition resulted in Cx43 up-regulation both in mRNA and protein levels, whereas AQP4 expression have not changed.  Discussion: In conclusion, data showed that fluoxetine alone and in the absence of serotonin acutely up-regulated Cx43 expression in astrocytes that can be assumed in molecular target therapy of MS patients. It seems that cAMP involvement in fluoxetine effects need more researches.

  8. Dlx homeobox gene family expression in osteoclasts.

    Science.gov (United States)

    Lézot, F; Thomas, B L; Blin-Wakkach, C; Castaneda, B; Bolanos, A; Hotton, D; Sharpe, P T; Heymann, D; Carles, G F; Grigoriadis, A E; Berdal, A

    2010-06-01

    Skeletal growth and homeostasis require the finely orchestrated secretion of mineralized tissue matrices by highly specialized cells, balanced with their degradation by osteoclasts. Time- and site-specific expression of Dlx and Msx homeobox genes in the cells secreting these matrices have been identified as important elements in the regulation of skeletal morphology. Such specific expression patterns have also been reported in osteoclasts for Msx genes. The aim of the present study was to establish the expression patterns of Dlx genes in osteoclasts and identify their function in regulating skeletal morphology. The expression patterns of all Dlx genes were examined during the whole osteoclastogenesis using different in vitro models. The results revealed that Dlx1 and Dlx2 are the only Dlx family members with a possible function in osteoclastogenesis as well as in mature osteoclasts. Dlx5 and Dlx6 were detected in the cultures but appear to be markers of monocytes and their derivatives. In vivo, Dlx2 expression in osteoclasts was examined using a Dlx2/LacZ transgenic mouse. Dlx2 is expressed in a subpopulation of osteoclasts in association with tooth, brain, nerve, and bone marrow volumetric growths. Altogether the present data suggest a role for Dlx2 in regulation of skeletal morphogenesis via functions within osteoclasts. (c) 2010 Wiley-Liss, Inc.

  9. Cyclin d1 expression in odontogenic cysts.

    Science.gov (United States)

    Taghavi, Nasim; Modabbernia, Shirin; Akbarzadeh, Alireza; Sajjadi, Samad

    2013-01-01

    In the present study expression of cyclin D1 in the epithelial lining of odontogenic keratocyst, radicular cyst, dentigerous cyst and glandular odontogenic cyst was investigated to compare proliferative activity in these lesions. Immunohistochemical staining of cyclin D1 on formalin-fixed, paraffin-embedded tissue sections of odontogenic keratocysts (n=23), dentigerous cysts (n=20), radicular cysts (n=20) and glandular odontogenic cysts (n=5) was performed by standard EnVision method. Then, slides were studied to evaluate the following parameters in epithelial lining of cysts: expression, expression pattern, staining intensity and localization of expression. The data analysis showed statistically significant difference in cyclin D1 expression in studied groups (p keratocysts, but difference was not statistically significant among groups respectively (p=0.204, 0.469). Considering expression localization, cyclin D1 positive cells in odontogenic keratocysts and dentigerous cysts were frequently confined in parabasal layer, different from radicular cysts and glandular odontogenic cysts. The difference was statistically significant (p keratocyst and the entire cystic epithelium of glandular odontogenic cysts comparing to dentigerous cysts and radicular cysts, implying the possible role of G1-S cell cycle phase disturbances in the aggressiveness of odontogenic keratocyst and glandular odontogenic cyst.

  10. Rho GTPase expression in human myeloid cells.

    Directory of Open Access Journals (Sweden)

    Suzanne F G van Helden

    Full Text Available Myeloid cells are critical for innate immunity and the initiation of adaptive immunity. Strict regulation of the adhesive and migratory behavior is essential for proper functioning of these cells. Rho GTPases are important regulators of adhesion and migration; however, it is unknown which Rho GTPases are expressed in different myeloid cells. Here, we use a qPCR-based approach to investigate Rho GTPase expression in myeloid cells.We found that the mRNAs encoding Cdc42, RhoQ, Rac1, Rac2, RhoA and RhoC are the most abundant. In addition, RhoG, RhoB, RhoF and RhoV are expressed at low levels or only in specific cell types. More differentiated cells along the monocyte-lineage display lower levels of Cdc42 and RhoV, while RhoC mRNA is more abundant. In addition, the Rho GTPase expression profile changes during dendritic cell maturation with Rac1 being upregulated and Rac2 downregulated. Finally, GM-CSF stimulation, during macrophage and osteoclast differentiation, leads to high expression of Rac2, while M-CSF induces high levels of RhoA, showing that these cytokines induce a distinct pattern. Our data uncover cell type specific modulation of the Rho GTPase expression profile in hematopoietic stem cells and in more differentiated cells of the myeloid lineage.

  11. Shadows alter facial expressions of Noh masks.

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    Nobuyuki Kawai

    Full Text Available BACKGROUND: A Noh mask, worn by expert actors during performance on the Japanese traditional Noh drama, conveys various emotional expressions despite its fixed physical properties. How does the mask change its expressions? Shadows change subtly during the actual Noh drama, which plays a key role in creating elusive artistic enchantment. We here describe evidence from two experiments regarding how attached shadows of the Noh masks influence the observers' recognition of the emotional expressions. METHODOLOGY/PRINCIPAL FINDINGS: In Experiment 1, neutral-faced Noh masks having the attached shadows of the happy/sad masks were recognized as bearing happy/sad expressions, respectively. This was true for all four types of masks each of which represented a character differing in sex and age, even though the original characteristics of the masks also greatly influenced the evaluation of emotions. Experiment 2 further revealed that frontal Noh mask images having shadows of upward/downward tilted masks were evaluated as sad/happy, respectively. This was consistent with outcomes from preceding studies using actually tilted Noh mask images. CONCLUSIONS/SIGNIFICANCE: Results from the two experiments concur that purely manipulating attached shadows of the different types of Noh masks significantly alters the emotion recognition. These findings go in line with the mysterious facial expressions observed in Western paintings, such as the elusive qualities of Mona Lisa's smile. They also agree with the aesthetic principle of Japanese traditional art "yugen (profound grace and subtlety", which highly appreciates subtle emotional expressions in the darkness.

  12. The predictive nature of transcript expression levels on protein expression in adult human brain.

    Science.gov (United States)

    Bauernfeind, Amy L; Babbitt, Courtney C

    2017-04-24

    Next generation sequencing methods are the gold standard for evaluating expression of the transcriptome. When determining the biological implications of such studies, the assumption is often made that transcript expression levels correspond to protein levels in a meaningful way. However, the strength of the overall correlation between transcript and protein expression is inconsistent, particularly in brain samples. Following high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses of adult human brain samples, we compared the correlation in the expression of transcripts and proteins that support various biological processes, molecular functions, and that are located in different areas of the cell. Although most categories of transcripts have extremely weak predictive value for the expression of their associated proteins (R 2 values of < 10%), transcripts coding for protein kinases and membrane-associated proteins, including those that are part of receptors or ion transporters, are among those that are most predictive of downstream protein expression levels. The predictive value of transcript expression for corresponding proteins is variable in human brain samples, reflecting the complex regulation of protein expression. However, we found that transcriptomic analyses are appropriate for assessing the expression levels of certain classes of proteins, including those that modify proteins, such as kinases and phosphatases, regulate metabolic and synaptic activity, or are associated with a cellular membrane. These findings can be used to guide the interpretation of gene expression results from primate brain samples.

  13. Differential expression of granulopoiesis related genes in neutrophil subsets distinguished by membrane expression of CD177

    DEFF Research Database (Denmark)

    Hu, Nan; Mora-Jensen, Helena; Theilgaard-Mønch, Kim

    2014-01-01

    OBJECTIVE: Differential gene expression in CD177+ and CD177- neutrophils was investigated, in order to detect possible differences in neutrophil function which could be related to the pathogenesis of ANCA-associated Vasculitides (AAV). METHODS: Neutrophils were isolated from healthy controls (HC......) with high, negative or bimodal CD177 expression, and sorted into CD177+ and CD177- subpopulations. Total RNA was screened for expression of 24,000 probes with Illumina Ref-8 Beadchips. Genes showing differential expression between CD177+ and CD177- subsets in microarray analysis were re-assessed using...... quantitative-PCR. CD177 expression on neutrophil precursors in bone marrow was analyzed using quantitative PCR and flowcytometry. RESULTS: The proportion of CD177+ cells increased during neutrophil maturation in bone marrow. Fold change analysis of gene expression profile of sorted CD177+ and CD177...

  14. cis sequence effects on gene expression

    Directory of Open Access Journals (Sweden)

    Jacobs Kevin

    2007-08-01

    Full Text Available Abstract Background Sequence and transcriptional variability within and between individuals are typically studied independently. The joint analysis of sequence and gene expression variation (genetical genomics provides insight into the role of linked sequence variation in the regulation of gene expression. We investigated the role of sequence variation in cis on gene expression (cis sequence effects in a group of genes commonly studied in cancer research in lymphoblastoid cell lines. We estimated the proportion of genes exhibiting cis sequence effects and the proportion of gene expression variation explained by cis sequence effects using three different analytical approaches, and compared our results to the literature. Results We generated gene expression profiling data at N = 697 candidate genes from N = 30 lymphoblastoid cell lines for this study and used available candidate gene resequencing data at N = 552 candidate genes to identify N = 30 candidate genes with sufficient variance in both datasets for the investigation of cis sequence effects. We used two additive models and the haplotype phylogeny scanning approach of Templeton (Tree Scanning to evaluate association between individual SNPs, all SNPs at a gene, and diplotypes, with log-transformed gene expression. SNPs and diplotypes at eight candidate genes exhibited statistically significant (p cis sequence effects in our study, respectively. Conclusion Based on analysis of our results and the extant literature, one in four genes exhibits significant cis sequence effects, and for these genes, about 30% of gene expression variation is accounted for by cis sequence variation. Despite diverse experimental approaches, the presence or absence of significant cis sequence effects is largely supported by previously published studies.

  15. Metallothionein gene expression in renal cell carcinoma

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    Deeksha Pal

    2014-01-01

    Full Text Available Introduction: Metallothioneins (MTs are a group of low-molecular weight, cysteine-rich proteins. In general, MT is known to modulate three fundamental processes: (1 the release of gaseous mediators such as hydroxyl radical or nitric oxide, (2 apoptosis and (3 the binding and exchange of heavy metals such as zinc, cadmium or copper. Previous studies have shown a positive correlation between the expression of MT with invasion, metastasis and poor prognosis in various cancers. Most of the previous studies primarily used immunohistochemistry to analyze localization of MT in renal cell carcinoma (RCC. No information is available on the gene expression of MT2A isoform in different types and grades of RCC. Materials and Methods: In the present study, total RNA was isolated from 38 histopathologically confirmed cases of RCC of different types and grades. Corresponding adjacent normal renal parenchyma was taken as control. Real-time polymerase chain reaction (RT PCR analysis was done for the MT2A gene expression using b-actin as an internal control. All statistical calculations were performed using SPSS software. Results: The MT2A gene expression was found to be significantly increased (P < 0.01 in clear cell RCC in comparison with the adjacent normal renal parenchyma. The expression of MT2A was two to three-fold higher in sarcomatoid RCC, whereas there was no change in papillary and collecting duct RCC. MT2A gene expression was significantly higher in lower grade (grades I and II, P < 0.05, while no change was observed in high-grade tumor (grade III and IV in comparison to adjacent normal renal tissue. Conclusion: The first report of the expression of MT2A in different types and grades of RCC and also these data further support the role of MT2A in tumorigenesis.

  16. Channel properties of Nax expressed in neurons.

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    Masahito Matsumoto

    Full Text Available Nax is a sodium-concentration ([Na+]-sensitive Na channel with a gating threshold of ~150 mM for extracellular [Na+] ([Na+]o in vitro. We previously reported that Nax was preferentially expressed in the glial cells of sensory circumventricular organs including the subfornical organ, and was involved in [Na+] sensing for the control of salt-intake behavior. Although Nax was also suggested to be expressed in the neurons of some brain regions including the amygdala and cerebral cortex, the channel properties of Nax have not yet been adequately characterized in neurons. We herein verified that Nax was expressed in neurons in the lateral amygdala of mice using an antibody that was newly generated against mouse Nax. To investigate the channel properties of Nax expressed in neurons, we established an inducible cell line of Nax using the mouse neuroblastoma cell line, Neuro-2a, which is endogenously devoid of the expression of Nax. Functional analyses of this cell line revealed that the [Na+]-sensitivity of Nax in neuronal cells was similar to that expressed in glial cells. The cation selectivity sequence of the Nax channel in cations was revealed to be Na+ ≈ Li+ > Rb+ > Cs+ for the first time. Furthermore, we demonstrated that Nax bound to postsynaptic density protein 95 (PSD95 through its PSD95/Disc-large/ZO-1 (PDZ-binding motif at the C-terminus in neurons. The interaction between Nax and PSD95 may be involved in promoting the surface expression of Nax channels because the depletion of endogenous PSD95 resulted in a decrease in Nax at the plasma membrane. These results indicated, for the first time, that Nax functions as a [Na+]-sensitive Na channel in neurons as well as in glial cells.

  17. LocExpress: a web server for efficiently estimating expression of novel transcripts.

    Science.gov (United States)

    Hou, Mei; Tian, Feng; Jiang, Shuai; Kong, Lei; Yang, Dechang; Gao, Ge

    2016-12-22

    The temporal and spatial-specific expression pattern of a transcript in multiple tissues and cell types can indicate key clues about its function. While several gene atlas available online as pre-computed databases for known gene models, it's still challenging to get expression profile for previously uncharacterized (i.e. novel) transcripts efficiently. Here we developed LocExpress, a web server for efficiently estimating expression of novel transcripts across multiple tissues and cell types in human (20 normal tissues/cells types and 14 cell lines) as well as in mouse (24 normal tissues/cell types and nine cell lines). As a wrapper to RNA-Seq quantification algorithm, LocExpress efficiently reduces the time cost by making abundance estimation calls increasingly within the minimum spanning bundle region of input transcripts. For a given novel gene model, such local context-oriented strategy allows LocExpress to estimate its FPKMs in hundreds of samples within minutes on a standard Linux box, making an online web server possible. To the best of our knowledge, LocExpress is the only web server to provide nearly real-time expression estimation for novel transcripts in common tissues and cell types. The server is publicly available at http://loc-express.cbi.pku.edu.cn .

  18. Morphing between expressions dissociates continuous from categorical representations of facial expression in the human brain

    Science.gov (United States)

    Harris, Richard J.; Young, Andrew W.; Andrews, Timothy J.

    2012-01-01

    Whether the brain represents facial expressions as perceptual continua or as emotion categories remains controversial. Here, we measured the neural response to morphed images to directly address how facial expressions of emotion are represented in the brain. We found that face-selective regions in the posterior superior temporal sulcus and the amygdala responded selectively to changes in facial expression, independent of changes in identity. We then asked whether the responses in these regions reflected categorical or continuous neural representations of facial expression. Participants viewed images from continua generated by morphing between faces posing different expressions such that the expression could be the same, could involve a physical change but convey the same emotion, or could differ by the same physical amount but be perceived as two different emotions. We found that the posterior superior temporal sulcus was equally sensitive to all changes in facial expression, consistent with a continuous representation. In contrast, the amygdala was only sensitive to changes in expression that altered the perceived emotion, demonstrating a more categorical representation. These results offer a resolution to the controversy about how facial expression is processed in the brain by showing that both continuous and categorical representations underlie our ability to extract this important social cue. PMID:23213218

  19. Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System

    NARCIS (Netherlands)

    Koenderink, J.B.; Swarts, H.G.

    2016-01-01

    P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed

  20. Gene expression analysis of flax seed development

    Science.gov (United States)

    2011-01-01

    Background Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. Results We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. Conclusions We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise

  1. Gene expression analysis of flax seed development

    Directory of Open Access Journals (Sweden)

    Sharpe Andrew

    2011-04-01

    Full Text Available Abstract Background Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. Results We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages seed coats (globular and torpedo stages and endosperm (pooled globular to torpedo stages and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST (GenBank accessions LIBEST_026995 to LIBEST_027011 were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152 had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. Conclusions We have developed a foundational database of expressed sequences and collection of plasmid

  2. Transgene expression in cowpea ( Vigna unguiculata (L.) Walp ...

    African Journals Online (AJOL)

    Transgene expression in cowpea ( Vigna unguiculata (L.) Walp.) ... and Bar genes for β-glucuronidase expression and bialaphos resistance respectively. ... expression also showed positive signals under PCR and Southern analysis giving ...

  3. Three new shuttle vectors for heterologous expression in Zymomonas mobilis

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    Qinghua Cao

    2016-01-01

    Conclusions: These results indicated that these expression vectors are useful tools for gene expression in Z. mobilis and this could provide a solid foundation for further studies of heterologous gene expression in Z. mobilis.

  4. Preventing, detecting & revising flaws in object property expressions

    CSIR Research Space (South Africa)

    Keet, CM

    2013-09-01

    Full Text Available The OWL 2 DL ontology language is very expressive and has many features for declaring complex object property expressions. Standard reasoning services for OWL ontologies take these expressions as correct and according to the ontologist's intention...

  5. Processing of emotional facial expressions in Korsakoff's syndrome.

    NARCIS (Netherlands)

    Montagne, B.; Kessels, R.P.C.; Wester, A.J.; Haan, E.H.F. de

    2006-01-01

    Interpersonal contacts depend to a large extent on understanding emotional facial expressions of others. Several neurological conditions may affect proficiency in emotional expression recognition. It has been shown that chronic alcoholics are impaired in labelling emotional expressions. More

  6. Time perception and dynamics of facial expressions of emotions.

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    Sophie L Fayolle

    Full Text Available Two experiments were run to examine the effects of dynamic displays of facial expressions of emotions on time judgments. The participants were given a temporal bisection task with emotional facial expressions presented in a dynamic or a static display. Two emotional facial expressions and a neutral expression were tested and compared. Each of the emotional expressions had the same affective valence (unpleasant, but one was high-arousing (expressing anger and the other low-arousing (expressing sadness. Our results showed that time judgments are highly sensitive to movements in facial expressions and the emotions expressed. Indeed, longer perceived durations were found in response to the dynamic faces and the high-arousing emotional expressions compared to the static faces and low-arousing expressions. In addition, the facial movements amplified the effect of emotions on time perception. Dynamic facial expressions are thus interesting tools for examining variations in temporal judgments in different social contexts.

  7. Decomposition of gene expression state space trajectories.

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    Jessica C Mar

    2009-12-01

    Full Text Available Representing and analyzing complex networks remains a roadblock to creating dynamic network models of biological processes and pathways. The study of cell fate transitions can reveal much about the transcriptional regulatory programs that underlie these phenotypic changes and give rise to the coordinated patterns in expression changes that we observe. The application of gene expression state space trajectories to capture cell fate transitions at the genome-wide level is one approach currently used in the literature. In this paper, we analyze the gene expression dataset of Huang et al. (2005 which follows the differentiation of promyelocytes into neutrophil-like cells in the presence of inducers dimethyl sulfoxide and all-trans retinoic acid. Huang et al. (2005 build on the work of Kauffman (2004 who raised the attractor hypothesis, stating that cells exist in an expression landscape and their expression trajectories converge towards attractive sites in this landscape. We propose an alternative interpretation that explains this convergent behavior by recognizing that there are two types of processes participating in these cell fate transitions-core processes that include the specific differentiation pathways of promyelocytes to neutrophils, and transient processes that capture those pathways and responses specific to the inducer. Using functional enrichment analyses, specific biological examples and an analysis of the trajectories and their core and transient components we provide a validation of our hypothesis using the Huang et al. (2005 dataset.

  8. A promoter-level mammalian expression atlas

    KAUST Repository

    Forest, Alistair R R

    2014-03-26

    Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

  9. Pragmatic expressions in cross-linguistic perspective

    Directory of Open Access Journals (Sweden)

    Maryann Overstreet

    2012-09-01

    Full Text Available This paper focuses on some pragmatic expressions that are characteristic of informal spoken English, their possible equivalents in some other languages, and their use by EFL learners from different backgrounds. These expressions, called general extenders (e.g. and stuff, or something, are shown to be different from discourse markers and to exhibit variation in form, function and distribution across varieties of English, as well as in other languages. In EFL contexts, students are reported to use fewer pragmatic expressions and a smaller range of possible forms. They also tend to favor expressions more often associated with writing and formal speaking (e.g. and so on, include literal translation equivalents from their first language that are not used in English (e.g. and, and, and, or used only in restricted contexts (e.g. or so, and often seem not to realize that some forms may carry negative connotations (e.g. and blah, blah, blah. The possibility of fostering better pragmatic awareness among EFL students is discussed in terms of an explicit cross-linguistic focus on the forms and functions of pragmatic expressions.

  10. The perception of emotion in body expressions.

    Science.gov (United States)

    de Gelder, B; de Borst, A W; Watson, R

    2015-01-01

    During communication, we perceive and express emotional information through many different channels, including facial expressions, prosody, body motion, and posture. Although historically the human body has been perceived primarily as a tool for actions, there is now increased understanding that the body is also an important medium for emotional expression. Indeed, research on emotional body language is rapidly emerging as a new field in cognitive and affective neuroscience. This article reviews how whole-body signals are processed and understood, at the behavioral and neural levels, with specific reference to their role in emotional communication. The first part of this review outlines brain regions and spectrotemporal dynamics underlying perception of isolated neutral and affective bodies, the second part details the contextual effects on body emotion recognition, and final part discusses body processing on a subconscious level. More specifically, research has shown that body expressions as compared with neutral bodies draw upon a larger network of regions responsible for action observation and preparation, emotion processing, body processing, and integrative processes. Results from neurotypical populations and masking paradigms suggest that subconscious processing of affective bodies relies on a specific subset of these regions. Moreover, recent evidence has shown that emotional information from the face, voice, and body all interact, with body motion and posture often highlighting and intensifying the emotion expressed in the face and voice. © 2014 John Wiley & Sons, Ltd.

  11. A validated battery of vocal emotional expressions

    Directory of Open Access Journals (Sweden)

    Pierre Maurage

    2007-11-01

    Full Text Available For a long time, the exploration of emotions focused on facial expression, and vocal expression of emotion has only recently received interest. However, no validated battery of emotional vocal expressions has been published and made available to the researchers’ community. This paper aims at validating and proposing such material. 20 actors (10 men recorded sounds (words and interjections expressing six basic emotions (anger, disgust, fear, happiness, neutral and sadness. These stimuli were then submitted to a double validation phase: (1 preselection by experts; (2 quantitative and qualitative validation by 70 participants. 195 stimuli were selected for the final battery, each one depicting a precise emotion. The ratings provide a complete measure of intensity and specificity for each stimulus. This paper provides, to our knowledge, the first validated, freely available and highly standardized battery of emotional vocal expressions (words and intonations. This battery could constitute an interesting tool for the exploration of prosody processing among normal and pathological populations, in neuropsychology as well as psychiatry. Further works are nevertheless needed to complement the present material.

  12. IL-21 Receptor Expression in Human Tendinopathy

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    Abigail L. Campbell

    2014-01-01

    Full Text Available The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy.

  13. A promoter-level mammalian expression atlas

    KAUST Repository

    Forest, Alistair R R; Kawaji, Hideya; Rehli, Michael; Baillie, John Kenneth; De Hoon, Michiel Jl L; Haberle, Vanja; Lassmann, Timo; Kulakovskiy, Ivan V.; Lizio, Marina; Itoh, Masayoshi; Andersson, Robin; Iida, Kei; Ikawa, Tomokatsu; Jankovic, Boris R.; Jia, Hui; Joshi, Anagha Madhusudan; Jurman, Giuseppe; Kaczkowski, Bogumił; Kai, Chieko; Kaida, Kaoru; Kaiho, Ai; Mungall, Christopher J.; Kajiyama, Kazuhiro; Kanamori-Katayama, Mutsumi; Kasianov, Artem S.; Kasukawa, Takeya; Katayama, Shintaro; Kato, Sachi; Kawaguchi, Shuji; Kawamoto, Hiroshi; Kawamura, Yuki I.; Kawashima, Tsugumi; Meehan, Terrence F.; Kempfle, Judith S.; Kenna, Tony J.; Kere, Juha; Khachigian, Levon M.; Kitamura, Toshio; Klinken, Svend Peter; Knox, Alan; Kojima, Miki; Kojima, Soichi; Kondo, Naoto; Schmeier, Sebastian; Koseki, Haruhiko; Koyasu, Shigeo; Krampitz, Sarah; Kubosaki, Atsutaka; Kwon, Andrew T.; Laros, Jeroen F J; Lee, Weonju; Lennartsson, Andreas; Li, Kang; Lilje, Berit; Bertin, Nicolas; Lipovich, Leonard; MacKay-Sim, Alan; Manabe, Riichiroh; Mar, Jessica; Marchand, Benoî t; Mathelier, Anthony; Mejhert, Niklas; Meynert, Alison M.; Mizuno, Yosuke; De Morais, David A Lima; Jø rgensen, Mette Christine; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Motakis, Efthymios; Motohashi, Hozumi; Mummery, Christine L.; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakachi, Yutaka; Nakahara, Fumio; Dimont, Emmanuel; Nakamura, Toshiyuki; Nakamura, Yukio; Nakazato, Kenichi; Van Nimwegen, Erik; Ninomiya, Noriko; Nishiyori, Hiromi; Noma, Shohei; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Arner, Erik; Ohmiya, Hiroko; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A.; Pain, Arnab; Passier, Robert C J J; Patrikakis, Margaret; Schmidl, Christian; Persson, Helena A.; Piazza, Silvano; Prendergast, James G D; Rackham, Owen J L; Ramilowski, Jordan A.; Rashid, Mamoon; Ravasi, Timothy; Rizzu, Patrizia; Roncador, Marco; Roy, Sugata; Schaefer, Ulf; Rye, Morten Beck; Saijyo, Eri; Sajantila, Antti; Saka, Akiko; Sakaguchi, Shimon; Sakai, Mizuho; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Medvedeva, Yulia; Schneider, Claudio H.; Schultes, Erik A.; Schulze-Tanzil, Gundula G.; Schwegmann, Anita; Sengstag, Thierry; Sheng, Guojun; Shimoji, Hisashi; Shimoni, Yishai; Shin, Jay W.; Simon, Chris M.; Plessy, Charles; Sugiyama, Daisuke; Sugiyama, Takaaki; Suzuki, Masanori; Suzuki, Naoko; Swoboda, Rolf K.; 't Hoen, Peter Ac Chr; Tagami, Michihira; Tagami, Naokotakahashi; Takai, Jun; Tanaka, Hiroshi; Vitezic, Morana; Tatsukawa, Hideki; Tatum, Zuotian; Thompson, Mark; Toyoda, Hiroo; Toyoda, Tetsuro; Valen, Eivind; Van De Wetering, Marc L.; Van Den Berg, Linda M.; Verardo, Roberto; Vijayan, Dipti; Severin, Jessica M.; Vorontsov, Ilya E.; Wasserman, Wyeth W.; Watanabe, Shoko; Wells, Christine A.; Winteringham, Louise Natalie; Wolvetang, Ernst Jurgen; Wood, Emily J.; Yamaguchi, Yoko; Yamamoto, Masayuki; Yoneda, Misako; Semple, Colin Am M; Yonekura, Yohei; Yoshida, Shigehiro; Zabierowski, Susan E.; Zhang, Peter; Zhao, Xiaobei; Zucchelli, Silvia; Summers, Kim M.; Suzuki, Harukazu; Daub, Carsten Olivier; Kawai, Jun; Ishizu, Yuri; Heutink, Peter; Hide, Winston; Freeman, Tom C.; Lenhard, Boris; Bajic, Vladimir B.; Taylor, Martin S.; Makeev, Vsevolod J.; Sandelin, Albin; Hume, David A.; Carninci, Piero; Young, Robert S.; Hayashizaki, Yoshihide Yoshihide; Francescatto, Margherita; Altschuler, Intikhab Alam; Albanese, Davide; Altschule, Gabriel M.; Arakawa, Takahiro; Archer, John A.C.; Arner, Peter; Babina, Magda; Rennie, Sarah; Balwierz, Piotr J.; Beckhouse, Anthony G.; Pradhan-Bhatt, Swati; Blake, Judith A.; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James A.; Brombacher, Frank; Burroughs, Alexander Maxwell; Califano, Andrea C.; Cannistraci, Carlo; Carbajo, Daniel; Chen, Yun; Chierici, Marco; Ciani, Yari; Clevers, Hans C.; Dalla, Emiliano; Davis, Carrie Anne; Detmar, Michael J.; Diehl, Alexander D.; Dohi, Taeko; Drablø s, Finn; Edge, Albert SB B; Edinger, Matthias G.; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Fagiolini, Michela; Fairbairn, Lynsey R.; Fang, Hai; Farach-Carson, Mary Cindy; Faulkner, Geoffrey J.; Favorov, Alexander V.; Fisher, Malcolm E.; Frith, Martin C.; Fujita, Rie; Fukuda, Shiro; Furlanello, Cesare; Furuno, Masaaki; Furusawa, Junichi; Geijtenbeek, Teunis Bh H; Gibson, Andrew P.; Gingeras, Thomas R.; Goldowitz, Dan; Gough, Julian; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas; Hamaguchi, Masahide; Hara, Mitsuko; Harbers, Matthias; Harshbarger, Jayson; Hasegawa, Akira; Hasegawa, Yuki; Hashimoto, Takehiro; Herlyn, Meenhard F.; Hitchens, Kelly J.; Sui, Shannan J Ho; Hofmann, Oliver M.; Hoof, Ilka; Hori, Fumi; Huminiecki, Łukasz B.

    2014-01-01

    Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

  14. Differential gene expression during Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Marco Aurelio Krieger

    1999-09-01

    Full Text Available The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE. The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells, while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.

  15. Developmental Hypothyroidism Reduces the Expression of ...

    Science.gov (United States)

    Disruption of thyroid hormone (TH) is a known effect of environmental contaminants. Neurotrophins including brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) have been implicated in brain dysfunction resulting from severe developmental TH insufficiency. Neurotrophins are also implicated in activity-dependent plasticity, a process critical for appropriate use-dependent connectivity in the developing brain and for memory formation in the adult. This study examined activity-induced expression of neurotrophin gene products in the hippocampus using the long-term potentiation (LTP) after developmental hypothyroidism induced by propylthiouracil (PTU). Pregnant rats were exposed to PTU (0 or I0ppm) via the drinking water from early gestation to weaning. Adult male offspring were anesthetized with urethane and implanted with electrodes in the dentate gyrus (00) and perforant path (PP). LTP was induced by PP stimulation and responses from 00 were monitored at 15m intervals until sacrifice of the animals 5 h later. The 00 was dissected from the stimulated and nonstimulated hemispheres for rtPCR analysis of the neurotrophins Bdnf, Ngf, Ntf3 and related genes Egrl, Arc, Klf9. We found no PTU-induced difference in basal levels of expression of any of these genes in the nonstimulated 00. LTP increased expression of Bdnf, Ngf, Arc and Klj9 in the control DG, and reduced expression of Ntf3. LTP in DG from PTU animals failed to increase expression of Bdnf,

  16. Mothers' pupillary responses to infant facial expressions.

    Science.gov (United States)

    Yrttiaho, Santeri; Niehaus, Dana; Thomas, Eileen; Leppänen, Jukka M

    2017-02-06

    Human parental care relies heavily on the ability to monitor and respond to a child's affective states. The current study examined pupil diameter as a potential physiological index of mothers' affective response to infant facial expressions. Pupillary time-series were measured from 86 mothers of young infants in response to an array of photographic infant faces falling into four emotive categories based on valence (positive vs. negative) and arousal (mild vs. strong). Pupil dilation was highly sensitive to the valence of facial expressions, being larger for negative vs. positive facial expressions. A separate control experiment with luminance-matched non-face stimuli indicated that the valence effect was specific to facial expressions and cannot be explained by luminance confounds. Pupil response was not sensitive to the arousal level of facial expressions. The results show the feasibility of using pupil diameter as a marker of mothers' affective responses to ecologically valid infant stimuli and point to a particularly prompt maternal response to infant distress cues.

  17. Evaluating Haskell expressions in a tutoring environment

    Directory of Open Access Journals (Sweden)

    Tim Olmer

    2014-12-01

    Full Text Available A number of introductory textbooks for Haskell use calculations right from the start to give the reader insight into the evaluation of expressions and the behavior of functional programs. Many programming concepts that are important in the functional programming paradigm, such as recursion, higher-order functions, pattern-matching, and lazy evaluation, can be partially explained by showing a stepwise computation. A student gets a better understanding of these concepts if she performs these evaluation steps herself. Tool support for experimenting with the evaluation of Haskell expressions is currently lacking. In this paper we present a prototype implementation of a stepwise evaluator for Haskell expressions that supports multiple evaluation strategies, specifically targeted at education. Besides performing evaluation steps the tool also diagnoses steps that are submitted by a student, and provides feedback. Instructors can add or change function definitions without knowledge of the tool's internal implementation. We discuss some preliminary results of a small survey about the tool.

  18. IER3 Expression in Childhood Myelodysplastic Syndrome

    DEFF Research Database (Denmark)

    de Vries, Andrica; Zwaan, Christian M.; Danen van Ooschot, Astrid

    Background: Childhood myelodysplastic syndrome (MDS) is a rare disease accounting for less than 5% of all hematological malignancies. In about 50% of the MDS cases an abnormal karyotype is found by conventional karyotyping, of which chromosome 6 is involved in 10%. The immediate-early-response 3...... (IER3) gene, which is located on chromosome 6p21, encodes for a glycoprotein that plays a role in the regulation of apoptosis and cell cycle progression. Recently, it was shown that IER3 gene aberrations frequently occur in adult MDS patients, which are not restricted to patients with chromosome 6...... aberrations and that low IER3 expression was associated with a worse outcome. Therefore, we investigated the frequency and prognostic impact of IER3 expression in childhood MDS. Methods: IER3 mRNA expression was determined by quantitative real-time PCR in 58 childhood MDS patients of which 17 carried...

  19. Greater perceptual sensitivity to happy facial expression.

    Science.gov (United States)

    Maher, Stephen; Ekstrom, Tor; Chen, Yue

    2014-01-01

    Perception of subtle facial expressions is essential for social functioning; yet it is unclear if human perceptual sensitivities differ in detecting varying types of facial emotions. Evidence diverges as to whether salient negative versus positive emotions (such as sadness versus happiness) are preferentially processed. Here, we measured perceptual thresholds for the detection of four types of emotion in faces--happiness, fear, anger, and sadness--using psychophysical methods. We also evaluated the association of the perceptual performances with facial morphological changes between neutral and respective emotion types. Human observers were highly sensitive to happiness compared with the other emotional expressions. Further, this heightened perceptual sensitivity to happy expressions can be attributed largely to the emotion-induced morphological change of a particular facial feature (end-lip raise).

  20. Drug-induced regulation of target expression

    DEFF Research Database (Denmark)

    Iskar, Murat; Campillos, Monica; Kuhn, Michael

    2010-01-01

    Drug perturbations of human cells lead to complex responses upon target binding. One of the known mechanisms is a (positive or negative) feedback loop that adjusts the expression level of the respective target protein. To quantify this mechanism systems-wide in an unbiased way, drug......-induced differential expression of drug target mRNA was examined in three cell lines using the Connectivity Map. To overcome various biases in this valuable resource, we have developed a computational normalization and scoring procedure that is applicable to gene expression recording upon heterogeneous drug treatments....... In 1290 drug-target relations, corresponding to 466 drugs acting on 167 drug targets studied, 8% of the targets are subject to regulation at the mRNA level. We confirmed systematically that in particular G-protein coupled receptors, when serving as known targets, are regulated upon drug treatment. We...

  1. Podocalyxin expression in malignant astrocytic tumors

    International Nuclear Information System (INIS)

    Hayatsu, Norihito; Kaneko, Mika Kato; Mishima, Kazuhiko; Nishikawa, Ryo; Matsutani, Masao; Price, Janet E.; Kato, Yukinari

    2008-01-01

    Podocalyxin is an anti-adhesive mucin-like transmembrane sialoglycoprotein that has been implicated in the development of aggressive forms of cancer. Podocalyxin is also known as keratan sulfate (KS) proteoglycan. Recently, we revealed that highly sulfated KS or another mucin-like transmembrane sialoglycoprotein podoplanin/aggrus is upregulated in malignant astrocytic tumors. The aim of this study is to examine the relationship between podocalyxin expression and malignant progression of astrocytic tumors. In this study, 51 astrocytic tumors were investigated for podocalyxin expression using immunohistochemistry, Western blot analysis, and quantitative real-time PCR. Immunohistochemistry detected podocalyxin on the surface of tumor cells in six of 14 anaplastic astrocytomas (42.9%) and in 17 of 31 glioblastomas (54.8%), especially around proliferating endothelial cells. In diffuse astrocytoma, podocalyxin expression was observed only in vascular endothelial cells. Podocalyxin might be associated with the malignant progression of astrocytic tumors, and be a useful prognostic marker for astrocytic tumors

  2. EXPRESSING DISTRESS IN PATIENTS WITH ADVANCED CANCER

    Directory of Open Access Journals (Sweden)

    Maura Gabriela FELEA

    2014-11-01

    Full Text Available Negative emotions (distress are recognized as part of the psychological profile of patients diagnosed with advanced stage cancer. However, most patients are not accustomed to verbalize feelings towards their physician, and generally towards family and medical care personnel. The purpose of this paper is to analyze the expression of emotions by patients in advanced stages of cancer, respectively the means by which they get to express emotions. To this respect, we identified the most common types of emotions expressed, or metaphors used by patients to describe their emotions and topics that trigger emotions. Words and phrases most commonly used are in relation to: fear, anxiety, depression, guilt, negligence, concern. They are uttered in order to depict the network created between disclosed emotions and topics on health status, symptoms, adverse effects and therapeutic choice, patient privacy, and social and family issues.

  3. Snail1 Expression Is Required for Sarcomagenesis

    Directory of Open Access Journals (Sweden)

    Lorena Alba-Castellón

    2014-05-01

    Full Text Available Snail1 transcriptional repressor is a major inducer of epithelial-to mesenchymal transition but is very limitedly expressed in adult animals. We have previously demonstrated that Snail1 is required for the maintenance of mesenchymal stem cells (MSCs, preventing their premature differentiation. Now, we show that Snail1 controls the tumorigenic properties of mesenchymal cells. Increased Snail1 expression provides tumorigenic capabilities to fibroblastic cells; on the contrary, Snail1 depletion decreases tumor growth. Genetic depletion of Snail1 in MSCs that are deficient in p53 tumor suppressor downregulates MSC markers and prevents the capability of these cells to originate sarcomas in immunodeficient SCID mice. Notably, an analysis of human sarcomas shows that, contrarily to epithelial tumors, these neoplasms display high Snail1 expression. This is particularly clear for undifferentiated tumors, which are associated with poor outcome. Together, our results indicate a role for Snail1 in the generation of sarcomas.

  4. Mental Health and Emotional Expression in Facebook

    Directory of Open Access Journals (Sweden)

    Eglee Duran Rodríguez

    2013-12-01

    Full Text Available The article reports the results of the project “Mental health and emotional expression in Facebook”. The research was approached from the qualitative paradigm under virtual ethnographic approach, interpreting the findings through their own players and triangulated with the views of researchers and experts in the area of mental health, emotions and information technology and communication. We concluded that a good part of users vented their secrets on Facebook, where they are able to confide and express a range of emotions and intimacies that in the real context is unlikely to give. Along these findings show that the use of Facebook serves as a space for emotional expression impacting the mental and emotional health.

  5. Structural development of child's artistic expression

    Directory of Open Access Journals (Sweden)

    Sanja Filipović

    2017-03-01

    Full Text Available Structural development implies control and capability of the expression usage in terms of independent creative expression and making. Understanding of structural development of child's artistic expression as a phenomenon (which is suitable to child's age has some implications on methodical acts considering the artistic education of children and youngsters. Therefore, it is of unexceptional importance to know these laws as well as methodical acts which encourage the structural development of artistic capabilities from an early age. Various experts dealt with this phenomenon, particularly Bogomil Karlavaris. In his methodical research, he has given an unexceptional part to this problem. It has been a starting point for analysis of certain methodical questions which are included in this work.

  6. Capsular Polysaccharide Expression in Commensal Streptococcus Species

    DEFF Research Database (Denmark)

    Skov Sørensen, Uffe B; Yao, Kaihu; Yang, Yonghong

    2016-01-01

    Expression of a capsular polysaccharide is considered a hallmark of most invasive species of bacteria, including Streptococcus pneumoniae, in which the capsule is among the principal virulence factors and is the basis for successful vaccines. Consequently, it was previously assumed that capsule....... pneumoniae evolved by import of cps fragments from commensal Streptococcus species, resulting in a mosaic of genes of different origins. The demonstrated antigenic identity of at least eight of the numerous capsular polysaccharide structures expressed by commensal streptococci with recognized serotypes of S...... of Streptococcus pneumoniae and is the basis for successful vaccines against infections caused by this important pathogen. Contrasting with previous assumptions, this study showed that expression of capsular polysaccharides by the same genetic mechanisms is a general property of closely related species...

  7. Brachyury expression in tailless Molgulid ascidian embryos.

    Science.gov (United States)

    Takada, Norio; York, Jonathan; Davis, J Muse; Schumpert, Brenda; Yasuo, Hitoyoshi; Satoh, Nori; Swalla, Billie J

    2002-01-01

    The T-box transcription factor gene Brachyury is important for the differentiation of notochord in all chordates, including the ascidians Halocynthia roretzi and Ciona intestinalis. We isolated Brachyury from molgulid ascidians, which have evolved tailless larvae multiple times independently, and found the genes appear functional by cDNA sequence analyses. We then compared the expression of Mocu-Bra in tailed Molgula oculata embryos to two tailless species, Molgula occulta (Mocc-Bra) and Molgula tectiformis (Mt-Bra). Here we show that both tailless species express Brachyury in the notochord lineage during embryogenesis. Initial expression of Mocu-Bra is normal in tailed M. oculata embryos; 10 precursor notochord cells divide twice to result in 40 notochord cells that converge and extend to make a notochord down the center of the tail. In contrast, in tailless Molgula occulta, Mocc-Bra expression disappears prematurely, and there is only one round of division, resulting in 20 cells in the final notochord lineage that never converge or extend. In M. occulta x M. oculata hybrid embryos, expression of Mocu-Bra is prolonged, and the embryos form a tail with 20 notochord cells that converge and extend normally. However, in Molgula tectiformis, a different tailless ascidian, Mt-Bra was expressed only in the 10 notochord precursor cells, which never divide, converge, or extend. In summary, neither Brachyury function nor the early establishment of the notochord lineage appears to be impaired in tailless embryos. In light of these results, we are continuing to investigate how and why notochord development is lost in tailless molgulid ascidian embryos.

  8. Population-expression models of immune response

    International Nuclear Information System (INIS)

    Stromberg, Sean P; Antia, Rustom; Nemenman, Ilya

    2013-01-01

    The immune response to a pathogen has two basic features. The first is the expansion of a few pathogen-specific cells to form a population large enough to control the pathogen. The second is the process of differentiation of cells from an initial naive phenotype to an effector phenotype which controls the pathogen, and subsequently to a memory phenotype that is maintained and responsible for long-term protection. The expansion and the differentiation have been considered largely independently. Changes in cell populations are typically described using ecologically based ordinary differential equation models. In contrast, differentiation of single cells is studied within systems biology and is frequently modeled by considering changes in gene and protein expression in individual cells. Recent advances in experimental systems biology make available for the first time data to allow the coupling of population and high dimensional expression data of immune cells during infections. Here we describe and develop population-expression models which integrate these two processes into systems biology on the multicellular level. When translated into mathematical equations, these models result in non-conservative, non-local advection-diffusion equations. We describe situations where the population-expression approach can make correct inference from data while previous modeling approaches based on common simplifying assumptions would fail. We also explore how model reduction techniques can be used to build population-expression models, minimizing the complexity of the model while keeping the essential features of the system. While we consider problems in immunology in this paper, we expect population-expression models to be more broadly applicable. (paper)

  9. Gene expression in periodontal tissues following treatment

    Directory of Open Access Journals (Sweden)

    Eisenacher Martin

    2008-07-01

    Full Text Available Abstract Background In periodontitis, treatment aimed at controlling the periodontal biofilm infection results in a resolution of the clinical and histological signs of inflammation. Although the cell types found in periodontal tissues following treatment have been well described, information on gene expression is limited to few candidate genes. Therefore, the aim of the study was to determine the expression profiles of immune and inflammatory genes in periodontal tissues from sites with severe chronic periodontitis following periodontal therapy in order to identify genes involved in tissue homeostasis. Gingival biopsies from 12 patients with severe chronic periodontitis were taken six to eight weeks following non-surgical periodontal therapy, and from 11 healthy controls. As internal standard, RNA of an immortalized human keratinocyte line (HaCaT was used. Total RNA was subjected to gene expression profiling using a commercially available microarray system focusing on inflammation-related genes. Post-hoc confirmation of selected genes was done by Realtime-PCR. Results Out of the 136 genes analyzed, the 5% most strongly expressed genes compared to healthy controls were Interleukin-12A (IL-12A, Versican (CSPG-2, Matrixmetalloproteinase-1 (MMP-1, Down syndrome critical region protein-1 (DSCR-1, Macrophage inflammatory protein-2β (Cxcl-3, Inhibitor of apoptosis protein-1 (BIRC-1, Cluster of differentiation antigen 38 (CD38, Regulator of G-protein signalling-1 (RGS-1, and Finkel-Biskis-Jinkins murine osteosarcoma virus oncogene (C-FOS; the 5% least strongly expressed genes were Receptor-interacting Serine/Threonine Kinase-2 (RIP-2, Complement component 3 (C3, Prostaglandin-endoperoxide synthase-2 (COX-2, Interleukin-8 (IL-8, Endothelin-1 (EDN-1, Plasminogen activator inhibitor type-2 (PAI-2, Matrix-metalloproteinase-14 (MMP-14, and Interferon regulating factor-7 (IRF-7. Conclusion Gene expression profiles found in periodontal tissues following

  10. Shadows Alter Facial Expressions of Noh Masks

    Science.gov (United States)

    Kawai, Nobuyuki; Miyata, Hiromitsu; Nishimura, Ritsuko; Okanoya, Kazuo

    2013-01-01

    Background A Noh mask, worn by expert actors during performance on the Japanese traditional Noh drama, conveys various emotional expressions despite its fixed physical properties. How does the mask change its expressions? Shadows change subtly during the actual Noh drama, which plays a key role in creating elusive artistic enchantment. We here describe evidence from two experiments regarding how attached shadows of the Noh masks influence the observers’ recognition of the emotional expressions. Methodology/Principal Findings In Experiment 1, neutral-faced Noh masks having the attached shadows of the happy/sad masks were recognized as bearing happy/sad expressions, respectively. This was true for all four types of masks each of which represented a character differing in sex and age, even though the original characteristics of the masks also greatly influenced the evaluation of emotions. Experiment 2 further revealed that frontal Noh mask images having shadows of upward/downward tilted masks were evaluated as sad/happy, respectively. This was consistent with outcomes from preceding studies using actually tilted Noh mask images. Conclusions/Significance Results from the two experiments concur that purely manipulating attached shadows of the different types of Noh masks significantly alters the emotion recognition. These findings go in line with the mysterious facial expressions observed in Western paintings, such as the elusive qualities of Mona Lisa’s smile. They also agree with the aesthetic principle of Japanese traditional art “yugen (profound grace and subtlety)”, which highly appreciates subtle emotional expressions in the darkness. PMID:23940748

  11. Covariance expressions for eigenvalue and eigenvector problems

    Science.gov (United States)

    Liounis, Andrew J.

    There are a number of important scientific and engineering problems whose solutions take the form of an eigenvalue--eigenvector problem. Some notable examples include solutions to linear systems of ordinary differential equations, controllability of linear systems, finite element analysis, chemical kinetics, fitting ellipses to noisy data, and optimal estimation of attitude from unit vectors. In many of these problems, having knowledge of the eigenvalue and eigenvector Jacobians is either necessary or is nearly as important as having the solution itself. For instance, Jacobians are necessary to find the uncertainty in a computed eigenvalue or eigenvector estimate. This uncertainty, which is usually represented as a covariance matrix, has been well studied for problems similar to the eigenvalue and eigenvector problem, such as singular value decomposition. There has been substantially less research on the covariance of an optimal estimate originating from an eigenvalue-eigenvector problem. In this thesis we develop two general expressions for the Jacobians of eigenvalues and eigenvectors with respect to the elements of their parent matrix. The expressions developed make use of only the parent matrix and the eigenvalue and eigenvector pair under consideration. In addition, they are applicable to any general matrix (including complex valued matrices, eigenvalues, and eigenvectors) as long as the eigenvalues are simple. Alongside this, we develop expressions that determine the uncertainty in a vector estimate obtained from an eigenvalue-eigenvector problem given the uncertainty of the terms of the matrix. The Jacobian expressions developed are numerically validated with forward finite, differencing and the covariance expressions are validated using Monte Carlo analysis. Finally, the results from this work are used to determine covariance expressions for a variety of estimation problem examples and are also applied to the design of a dynamical system.

  12. Human Eosinophils Express Functional CCR7

    Science.gov (United States)

    Ueki, Shigeharu; Estanislau, Jessica; Weller, Peter F.

    2013-01-01

    Human eosinophils display directed chemotactic activity toward an array of soluble chemokines. Eosinophils have been observed to migrate to draining lymph nodes in experimental models of allergic inflammation, yet it is unknown whether eosinophils express CCR7, a key chemokine receptor in coordinating leukocyte trafficking to lymph nodes. The purpose of this study is to demonstrate expression of CCR7 by human eosinophils and functional responses to CCL19 and CCL21, the known ligands of CCR7. Human eosinophils were purified by negative selection from healthy donors. CCR7 expression of freshly purified, unstimulated eosinophils and of IL-5–primed eosinophils was determined by flow cytometry and Western blot. Chemotaxis to CCL19 and CCL21 was measured in transwell assays. Shape changes to CCL19 and CCL21 were analyzed by flow cytometry and microscopy. Calcium fluxes of fluo-4 AM–loaded eosinophils were recorded by flow cytometry after chemokine stimulation. ERK phosphorylation of CCL19- and CCL21-stimulated eosinophils was measured by Western blot and Luminex assay. Human eosinophils expressed CCR7 as demonstrated by flow cytometry and Western blots. Eosinophils exhibited detectable cell surface expression of CCR7. IL-5–primed eosinophils exhibited chemotaxis toward CCL19 and CCL21 in a dose-dependent fashion. Upon stimulation with CCL19 or CCL21, IL-5–primed eosinophils demonstrated dose-dependent shape changes with polarization of F-actin and exhibited calcium influxes. Finally, primed eosinophils stimulated with CCL19 or CCL21 exhibited increased phosphorylation of ERK in response to both CCR7 ligands. We demonstrate that human eosinophils express CCR7 and have multipotent responses to the known ligands of CCR7. PMID:23449735

  13. Expert Oracle Application Express Plug-Ins

    CERN Document Server

    D'Souza, Martin Giffy

    2011-01-01

    Expert Oracle Application Express Plugins is your "go to" book on the groundbreaking plugin architecture introduced in Oracle Application Express 4.0. Using the new APEX functionality, you can create well-packaged, documented, reusable components and reliably leverage your coding investments across many applications. Components you create can define new item and region types, specify validation processes, and present dynamic actions to client applications. You can design innovative and colorful ways to display information, such as displaying the temperature using an image of a thermometer, or

  14. MHC class II expression in lung cancer.

    Science.gov (United States)

    He, Yayi; Rozeboom, Leslie; Rivard, Christopher J; Ellison, Kim; Dziadziuszko, Rafal; Yu, Hui; Zhou, Caicun; Hirsch, Fred R

    2017-10-01

    Immunotherapy is an exciting development in lung cancer research. In this study we described major histocompatibility complex (MHC) Class II protein expression in lung cancer cell lines and patient tissues. We studied MHC Class II (DP, DQ, DR) (CR3/43, Abcam) protein expression in 55 non-small cell lung cancer (NSCLC) cell lines, 42 small cell lung cancer (SCLC) cell lines and 278 lung cancer patient tissues by immunohistochemistry (IHC). Seven (12.7%) NSCLC cell lines were positive for MHC Class II. No SCLC cell lines were found to be MHC Class II positive. We assessed 139 lung cancer samples available in the Hirsch Lab for MHC Class II. There was no positive MHC Class II staining on SCLC tumor cells. MHC Class II expression on TILs in SCLC was significantly lower than that on TILs in NSCLC (P<0.001). MHC Class II was also assessed in an additional 139 NSCLC tumor tissues from Medical University of Gdansk, Poland. Patients with positive staining of MHC Class II on TILs had longer regression-free survival (RFS) and overall survival (OS) than those whose TILs were MHC Class II negative (2.980 years, 95% CI 1.628-4.332 vs. 1.050 years, 95% CI 0.556-1.554, P=0.028) (3.230 years, 95% CI 2.617-3.843 vs. 1.390 years, 95% CI 0.629-2.151, P=0.014). MHC Class II was expressed both in NSCLC cell lines and tissues. However, MHC Class II was not detected in SCLC cell lines or tissue tumor cells. MHC Class II expression was lower on SCLC TILs than on NSCLC TILs. Loss of expression of MHC Class II on SCLC tumor cells and reduced expression on SCLC TILs may be a means of escaping anti-cancer immunity. Higher MHC Class II expression on TILs was correlated with better prognosis in patients with NSCLC. Copyright © 2017. Published by Elsevier B.V.

  15. Parts Characterization for Tunable Protein Expression

    DEFF Research Database (Denmark)

    Klausen, Michael Schantz; Sommer, Morten Otto Alexander

    2018-01-01

    Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression. Construc......Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression...

  16. Assigning Numerical Scores to Linguistic Expressions

    Directory of Open Access Journals (Sweden)

    María Jesús Campión

    2017-07-01

    Full Text Available In this paper, we study different methods of scoring linguistic expressions defined on a finite set, in the search for a linear order that ranks all those possible expressions. Among them, particular attention is paid to the canonical extension, and its representability through distances in a graph plus some suitable penalization of imprecision. The relationship between this setting and the classical problems of numerical representability of orderings, as well as extension of orderings from a set to a superset is also explored. Finally, aggregation procedures of qualitative rankings and scorings are also analyzed.

  17. Encapsulated social perception of emotional expressions.

    Science.gov (United States)

    Smortchkova, Joulia

    2017-01-01

    In this paper I argue that the detection of emotional expressions is, in its early stages, informationally encapsulated. I clarify and defend such a view via the appeal to data from social perception on the visual processing of faces, bodies, facial and bodily expressions. Encapsulated social perception might exist alongside processes that are cognitively penetrated, and that have to do with recognition and categorization, and play a central evolutionary function in preparing early and rapid responses to the emotional stimuli. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Oncomirs Expression Profiling in Uterine Leiomyosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Bruna Cristine de Almeida

    2017-12-01

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs that act as regulators of gene expression at the post-transcriptional level. They play a key role in several biological processes. Their abnormal expression may lead to malignant cell transformation. This study aimed to evaluate the expression profile of 84 miRNAs involved in tumorigenesis in immortalized cells of myometrium (MM, uterine leiomyoma (ULM, and uterine leiomyosarcoma (ULMS. Specific cell lines were cultured and qRT-PCR was performed. Thirteen miRNAs presented different expression profiles in ULM and the same thirteen in ULMS compared to MM. Eight miRNAs were overexpressed, and five were underexpressed in ULM. In ULMS cells, five miRNAs exhibited an overexpression and eight were down-regulated. Six miRNAs (miR-1-3p, miR-130b-3p, miR-140-5p, miR-202-3p, miR-205-5p, and miR-7-5p presented a similar expression pattern in cell lines compared to patient samples. Of these, only three miRNAs showed significant expression in ULM (miR-1-3p, miR-140-5p, and miR-7-5p and ULMS (miR-1-3p, miR-202-3p, and miR-7-5p. Our preliminary approach identified 24 oncomirs with an altered expression profile in ULM and ULMS cells. We identified four differentially expressed miRNAs with the same profile when compared with patients’ samples, which strongly interacted with relevant genes, including apoptosis regulator (BCL2, epidermal growth factor receptor (EGFR, vascular endothelial growth factor A (VEGFA, insulin like growth factor 1 receptor (IGF1R,serine/threonine kinase (RAF1, receptor tyrosine kinase (MET, and bHLH transcription factor (MYCN. This led to alterations in their mRNA-target.

  19. MEMBRANE LEc EXPRESSION IN BREAST CANCER CELLS

    Directory of Open Access Journals (Sweden)

    Ya. A. Udalova

    2009-01-01

    Full Text Available Affine chromatography was used to isolate Lec antibodies from the sera of a healthy female donor with the high titers of these anti- bodies, which were labeled with biotin. The study enrolled 51 patients with primary breast cancer (BC. Antigen expression was found by immunohistochemistry and flow cytometry. With these two techniques being used, the detection rate of Lec expression in BC cells was 65% (33/51; the antigen was most frequently found by flow cytometry as compared with immunohistochemistry: 72 and 58% of cases, respectively.

  20. Comparative gene expression of intestinal metabolizing enzymes.

    Science.gov (United States)

    Shin, Ho-Chul; Kim, Hye-Ryoung; Cho, Hee-Jung; Yi, Hee; Cho, Soo-Min; Lee, Dong-Goo; Abd El-Aty, A M; Kim, Jin-Suk; Sun, Duxin; Amidon, Gordon L

    2009-11-01

    The purpose of this study was to compare the expression profiles of drug-metabolizing enzymes in the intestine of mouse, rat and human. Total RNA was isolated from the duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mouse, rat and human were ca. 60% of 22690 sequences, 40% of 8739 and 47% of 12559, respectively. Total genes of metabolizing enzymes subjected in this study were 95, 33 and 68 genes in mouse, rat and human, respectively. Of phase I enzymes, the mouse exhibited abundant gene expressions for Cyp3a25, Cyp4v3, Cyp2d26, followed by Cyp2b20, Cyp2c65 and Cyp4f14, whereas, the rat showed higher expression profiles of Cyp3a9, Cyp2b19, Cyp4f1, Cyp17a1, Cyp2d18, Cyp27a1 and Cyp4f6. However, the highly expressed P450 enzymes were CYP3A4, CYP3A5, CYP4F3, CYP2C18, CYP2C9, CYP2D6, CYP3A7, CYP11B1 and CYP2B6 in the human. For phase II enzymes, glucuronosyltransferase Ugt1a6, glutathione S-transferases Gstp1, Gstm3 and Gsta2, sulfotransferase Sult1b1 and acyltransferase Dgat1 were highly expressed in the mouse. The rat revealed predominant expression of glucuronosyltransferases Ugt1a1 and Ugt1a7, sulfotransferase Sult1b1, acetyltransferase Dlat and acyltransferase Dgat1. On the other hand, in human, glucuronosyltransferases UGT2B15 and UGT2B17, glutathione S-transferases MGST3, GSTP1, GSTA2 and GSTM4, sulfotransferases ST1A3 and SULT1A2, acetyltransferases SAT1 and CRAT, and acyltransferase AGPAT2 were dominantly detected. Therefore, current data indicated substantial interspecies differences in the pattern of intestinal gene expression both for P450 enzymes and phase II drug-metabolizing enzymes. This genomic database is expected to improve our understanding of interspecies variations in estimating intestinal prehepatic clearance of oral drugs.