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Sample records for cell selection revealed

  1. Quantification of uncoupling protein 2 reveals its main expression in immune cells and selective up-regulation during T-cell proliferation.

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    Anne Rupprecht

    Full Text Available Uncoupling protein 2 (UCP2 is an inner mitochondrial membrane protein. Although the protein was discovered in 1997, its function and even its tissue distribution are still under debate. Here we present a quantitative analysis of mRNA and protein expression in various mice tissues, revealing that UCP2 is mainly expressed in organs and cells associated with the immune system. Although the UCP2 gene is present in the brain, as demonstrated using quantitative RT-PCR, the protein was not detectable in neurons under physiological conditions. Instead, we could detect UCP2 in microglia, which act in the immune defense of the central nervous system. In lymphocytes, activation led to a ten-fold increase of UCP2 protein expression simultaneously to the increase in levels of other mitochondrial proteins, whereas lymphocyte re-stimulation resulted in the selective increase of UCP2. The highest detected level of UCP2 expression in stimulated T-cells (0.54 ng/(µg total cellular protein was approximately 200 times lower than the level of UCP1 in brown adipose tissue from room temperature acclimated mice. Both the UCP2 expression pattern and the time course of up-regulation in stimulated T-cells imply UCP2's involvement in the immune response, probably by controlling the metabolism during cell proliferation.

  2. Pim-selective inhibitor DHPCC-9 reveals Pim kinases as potent stimulators of cancer cell migration and invasion

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    Prudhomme Michelle

    2010-10-01

    Full Text Available Abstract Background Pim family kinases are small constitutively active serine/threonine-specific kinases, elevated levels of which have been detected in human hematopoietic malignancies as well as in solid tumours. While we and others have previously shown that the oncogenic Pim kinases stimulate survival of hematopoietic cells, we now examined their putative role in regulating motility of adherent cancer cells. For this purpose, we inhibited Pim kinase activity using a small molecule compound, 1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (DHPCC-9, which we had recently identified as a potent and selective inhibitor for all Pim family members. Results We now demonstrate that the Pim kinase inhibitor DHPCC-9 is very effective also in cell-based assays. DHPCC-9 impairs the anti-apoptotic effects of Pim-1 in cytokine-deprived myeloid cells and inhibits intracellular phosphorylation of Pim substrates such as Bad. Moreover, DHPCC-9 slows down migration and invasion of cancer cells derived from either prostate cancer or squamocellular carcinoma patients. Silencing of Pim expression reduces cell motility, while Pim overexpression enhances it, strongly suggesting that the observed effects of DHPCC-9 are dependent on Pim kinase activity. Interestingly, DHPCC-9 also abrogates NFATc-dependent migration of cancer cells, implying that NFATc factors mediate at least part of the pro-migratory effects of Pim kinases. Conclusions Altogether, our data indicate that DHPCC-9 is not only a powerful tool to investigate physiological effects of the oncogenic Pim family kinases, but also an attractive molecule for drug development to inhibit invasiveness of Pim-overexpressing cancer cells.

  3. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

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    Ruben R Bender

    2016-06-01

    Full Text Available Receptor-targeted lentiviral vectors (LVs can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance. Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4 was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

  4. Comparative analysis of a large panel of non-starch polysaccharides reveals structures with selective regulatory properties in dendritic cells

    DEFF Research Database (Denmark)

    Wismar, René; Pedersen, Susanne Brix; Lærke, Helle Nygaard;

    2011-01-01

    Scope: Structural-based recognition of foreign molecules is essential for activation of dendritic cells (DCs) that play a key role in regulation of gut mucosal immunity. Orally ingested non-starch polysaccharides (NSP) are ascribed many health-promoting properties, but currently we lack insight i...

  5. Genome-wide analysis reveals selective modulation of microRNAs and mRNAs by histone deacetylase inhibitor in B cells induced to undergo class switch DNA recombination and plasma cell differentiation

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    Tian eShen

    2015-12-01

    Full Text Available As we have suggested, epigenetic factors, such as microRNAs (miRNAs, can interact with genetic programs to regulate B cell functions, thereby informing antibody and autoantibody responses. We have shown that histone deacetylase inhibitors (HDI inhibit the differentiation events critical to the maturation of the antibody response: class-switch DNA recombination (CSR, somatic hypermutation (SHM and plasma cell differentiation, by modulating intrinsic B cell mechanisms. HDI repress the expression of AID and Blimp-1, which are critical for CSR/SHM and plasma cell differentiation, respectively, in mouse and human B cells by upregulating selected miRNAs that silenced AICDA/Aicda and PRDM1/Prdm1 mRNAs, as demonstrated by multiple qRT-PCRs (J. Immunol. 193:5933-5950, 2014. To further define the selectivity of HDI-mediated modulation of miRNA and gene expression, we performed genome-wide miRNA-Seq and mRNA-Seq analysis in B cells stimulated by LPS plus IL-4 and treated with HDI or nil. Consistent with what we have shown using qRT-PCR, these HDI-treated B cells displayed reduced expression of Aicda and Prdm1, and increased expression of miR-155, miR-181b and miR-361, which target Aicda, and miR-23b, miR-30a and miR-125b, which target Prdm1. In B cells induced to undergo CSR and plasma cell differentiation, about 23% of over 22,000 mRNAs analyzed were expressed at a significantly high copy number (more than 20 copies/cell. Only 18 (0.36% of these highly expressed mRNAs, including Aicda, Prdm1 and Xbp1, were downregulated by HDI by 50% or more. Further, only 16 (0.30% of the highly expressed mRNAs were upregulated (more than twofold by HDI. The selectivity of HDI-mediated modulation of gene expression was emphasized by unchanged expression of the genes that are involved in regulation, targeting or DNA repair processes of CSR, as well as unchanged expression of the genes encoding epigenetic regulators and factors that are important for cell signaling or

  6. Genome-Wide Analysis Reveals Selective Modulation of microRNAs and mRNAs by Histone Deacetylase Inhibitor in B Cells Induced to Undergo Class-Switch DNA Recombination and Plasma Cell Differentiation.

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    Shen, Tian; Sanchez, Helia N; Zan, Hong; Casali, Paolo

    2015-01-01

    As we have suggested, epigenetic factors, such as microRNAs (miRNAs), can interact with genetic programs to regulate B cell functions, thereby informing antibody and autoantibody responses. We have shown that histone deacetylase (HDAC) inhibitors (HDI) inhibit the differentiation events critical to the maturation of the antibody response: class-switch DNA recombination (CSR), somatic hypermutation (SHM), and plasma cell differentiation, by modulating intrinsic B cell mechanisms. HDI repress the expression of AID and Blimp-1, which are critical for CSR/SHM and plasma cell differentiation, respectively, in mouse and human B cells by upregulating selected miRNAs that silenced AICDA/Aicda and PRDM1/Prdm1 mRNAs, as demonstrated by multiple qRT-PCRs (J Immunol 193:5933-5950, 2014). To further define the selectivity of HDI-mediated modulation of miRNA and gene expression, we performed genome-wide miRNA-Seq and mRNA-Seq analysis in B cells stimulated by LPS plus IL-4 and treated with HDI or nil. Consistent with what we have shown using qRT-PCR, these HDI-treated B cells displayed reduced expression of Aicda and Prdm1, and increased expression of miR-155, miR-181b, and miR-361, which target Aicda, and miR-23b, miR-30a, and miR-125b, which target Prdm1. In B cells induced to undergo CSR and plasma cell differentiation, about 23% of over 22,000 mRNAs analyzed were expressed at a significantly high copy number (more than 20 copies/cell). Only 18 (0.36%) of these highly expressed mRNAs, including Aicda, Prdm1, and Xbp1, were downregulated by HDI by 50% or more. Further, only 16 (0.30%) of the highly expressed mRNAs were upregulated (more than twofold) by HDI. The selectivity of HDI-mediated modulation of gene expression was emphasized by unchanged expression of the genes that are involved in regulation, targeting, or DNA repair processes of CSR, as well as unchanged expression of the genes encoding epigenetic regulators and factors that are important for cell signaling or

  7. Molecular profiling of blastic plasmacytoid dendritic cell neoplasm reveals a unique pattern and suggests selective sensitivity to NF-kB pathway inhibition

    Science.gov (United States)

    Sapienza, MR; Fuligni, F; Agostinelli, C; Tripodo, C; Righi, S; Laginestra, MA; Pileri, A; Mancini, M; Rossi, M; Ricci, F; Gazzola, A; Melle, F; Mannu, C; Ulbar, F; Arpinati, M; Paulli, M; Maeda, T; Gibellini, D; Pagano, L; Pimpinelli, N; Santucci, M; Cerroni, L; Croce, CM; Facchetti, F; Piccaluga, PP; Pileri, SA

    2015-01-01

    Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare disease of controversial origin recently recognized as a neoplasm deriving from plasmacytoid dendritic cells (pDCs). Nevertheless, it remains an orphan tumor with obscure biology and dismal prognosis. To better understand the pathobiology of BPDCN and discover new targets for effective therapies, the gene expression profile (GEP) of 25 BPDCN samples was analyzed and compared with that of pDCs, their postulated normal counterpart. Validation was performed by immunohistochemistry (IHC), whereas functional experiments were carried out ex vivo. For the first time at the molecular level, we definitely recognized the cellular derivation of BPDCN that proved to originate from the myeloid lineage and in particular, from resting pDCs. Furthermore, thanks to an integrated bioinformatic approach we discovered aberrant activation of the NF-kB pathway and suggested it as a novel therapeutic target. We tested the efficacy of anti-NF-kB-treatment on the BPDCN cell line CAL-1, and successfully demonstrated by GEP and IHC the molecular shutoff of the NF-kB pathway. In conclusion, we identified a molecular signature representative of the transcriptional abnormalities of BPDCN and developed a cellular model proposing a novel therapeutic approach in the setting of this otherwise incurable disease. PMID:24504027

  8. Parallel Selection Revealed by Population Sequencing in Chicken.

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    Qanbari, Saber; Seidel, Michael; Strom, Tim-Mathias; Mayer, Klaus F X; Preisinger, Ruedi; Simianer, Henner

    2015-12-01

    Human-driven selection during domestication and subsequent breed formation has likely left detectable signatures within the genome of modern chicken. The elucidation of these signatures of selection is of interest from the perspective of evolutionary biology, and for identifying genes relevant to domestication and improvement that ultimately may help to further genetically improve this economically important animal. We used whole genome sequence data from 50 hens of commercial white (WL) and brown (BL) egg-laying chicken along with pool sequences of three meat-type chicken to perform a systematic screening of past selection in modern chicken. Evidence of positive selection was investigated in two steps. First, we explored evidence of parallel fixation in regions with overlapping elevated allele frequencies in replicated populations of layers and broilers, suggestive of selection during domestication or preimprovement ages. We confirmed parallel fixation in BCDO2 and TSHR genes and found four candidates including AGTR2, a gene heavily involved in "Ascites" in commercial birds. Next, we explored differentiated loci between layers and broilers suggestive of selection during improvement in chicken. This analysis revealed evidence of parallel differentiation in genes relevant to appearance and production traits exemplified with the candidate gene OPG, implicated in Osteoporosis, a disorder related to overconsumption of calcium in egg-laying hens. Our results illustrate the potential for population genetic techniques to identify genomic regions relevant to the phenotypes of importance to breeders. PMID:26568375

  9. Early allelic selection in maize as revealed by ancient DNA.

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    Jaenicke-Després, Viviane; Buckler, Ed S; Smith, Bruce D; Gilbert, M Thomas P; Cooper, Alan; Doebley, John; Pääbo, Svante

    2003-11-14

    Maize was domesticated from teosinte, a wild grass, by approximately 6300 years ago in Mexico. After initial domestication, early farmers continued to select for advantageous morphological and biochemical traits in this important crop. However, the timing and sequence of character selection are, thus far, known only for morphological features discernible in corn cobs. We have analyzed three genes involved in the control of plant architecture, storage protein synthesis, and starch production from archaeological maize samples from Mexico and the southwestern United States. The results reveal that the alleles typical of contemporary maize were present in Mexican maize by 4400 years ago. However, as recently as 2000 years ago, allelic selection at one of the genes may not yet have been complete. PMID:14615538

  10. A retinoid X receptor (RXR)-selective retinoid reveals that RXR-α is potentially a therapeutic target in breast cancer cell lines, and that it potentiates antiproliferative and apoptotic responses to peroxisome proliferator-activated receptor ligands

    International Nuclear Information System (INIS)

    Certain lipids have been shown to be ligands for a subgroup of the nuclear hormone receptor superfamily known as the peroxisome proliferator-activated receptors (PPARs). Ligands for these transcription factors have been used in experimental cancer therapies. PPARs heterodimerize and bind DNA with retinoid X receptors (RXRs), which have homology to other members of the nuclear receptor superfamily. Retinoids have been found to be effective in treating many types of cancer. However, many breast cancers become resistant to the chemotherapeutic effects of these drugs. Recently, RXR-selective ligands were discovered that inhibited proliferation of all-trans retinoic acid resistant breast cancer cells in vitro and caused regression of the disease in animal models. There are few published studies on the efficacy of combined therapy using PPAR and RXR ligands for breast cancer prevention or treatment. We determined the effects of selective PPAR and RXR ligands on established human breast cancer cell lines in vitro. PPAR-α and PPAR-γ ligands induced apoptotic and antiproliferative responses in human breast cancer cell lines, respectively, which were associated with specific changes in gene expression. These responses were potentiated by the RXR-selective ligand AGN194204. Interestingly, RXR-α-overexpressing retinoic acid resistant breast cancer cell lines were more sensitive to the effects of the RXR-selective compound. RXR-selective retinoids can potentiate the antiproliferative and apoptotic responses of breast cancer cell lines to PPAR ligands

  11. Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes.

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    Wu, I; Shin, S C; Cao, Y; Bender, I K; Jafari, N; Feng, G; Lin, S; Cidlowski, J A; Schleimer, R P; Lu, N Z

    2013-01-01

    Induction of T-cell apoptosis contributes to the anti-inflammatory and antineoplastic benefits of glucocorticoids. The glucocorticoid receptor (GR) translational isoforms have distinct proapoptotic activities in osteosarcoma cells. Here we determined whether GR isoforms selectively induce apoptosis in Jurkat T lymphoblastic leukemia cells. Jurkat cells stably expressing individual GR isoforms were generated and treated with vehicle or dexamethasone (DEX). DEX induced apoptosis in cells expressing the GR-A, -B, or -C, but not the GR-D, isoform. cDNA microarray analyses of cells sensitive (GR-C3) and insensitive (GR-D3) to DEX revealed glucocorticoid-induced proapoptotic transcriptomes. Genes that were regulated by the proapoptotic GR-C3, but not by the GR-D3, isoform likely contributed to glucocorticoid-induced apoptosis. The identified genes include those that are directly involved in apoptosis and those that facilitate cell killing. Chromatin immunoprecipitation assays demonstrated that distinct chromatin modification abilities may underlie the distinct functions of GR isoforms. Interestingly, all GR isoforms, including the GR-D3 isoform, suppressed mitogen-stimulated cytokines. Furthermore, the GR-C isoforms were selectively upregulated in mitogen-activated primary T cells and DEX treatment induced GR-C target genes in activated T cells. Cell-specific expressions and functions of GR isoforms may help to explain the tissue- and individual-selective actions of glucocorticoids and may provide a basis for developing improved glucocorticoids. PMID:23303127

  12. Single cell transcriptional analysis reveals novel innate immune cell types

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    Linda E. Kippner

    2014-06-01

    Full Text Available Single-cell analysis has the potential to provide us with a host of new knowledge about biological systems, but it comes with the challenge of correctly interpreting the biological information. While emerging techniques have made it possible to measure inter-cellular variability at the transcriptome level, no consensus yet exists on the most appropriate method of data analysis of such single cell data. Methods for analysis of transcriptional data at the population level are well established but are not well suited to single cell analysis due to their dependence on population averages. In order to address this question, we have systematically tested combinations of methods for primary data analysis on single cell transcription data generated from two types of primary immune cells, neutrophils and T lymphocytes. Cells were obtained from healthy individuals, and single cell transcript expression data was obtained by a combination of single cell sorting and nanoscale quantitative real time PCR (qRT-PCR for markers of cell type, intracellular signaling, and immune functionality. Gene expression analysis was focused on hierarchical clustering to determine the existence of cellular subgroups within the populations. Nine combinations of criteria for data exclusion and normalization were tested and evaluated. Bimodality in gene expression indicated the presence of cellular subgroups which were also revealed by data clustering. We observed evidence for two clearly defined cellular subtypes in the neutrophil populations and at least two in the T lymphocyte populations. When normalizing the data by different methods, we observed varying outcomes with corresponding interpretations of the biological characteristics of the cell populations. Normalization of the data by linear standardization taking into account technical effects such as plate effects, resulted in interpretations that most closely matched biological expectations. Single cell transcription

  13. Genomic signatures reveal geographic adaption and human selection in cattle

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    We investigated geographic adaptation and human selection using high-density SNP data of five diverse cattle breeds. Based on allele frequency differences, we detected hundreds of candidate regions under positive selection across Holstein, Angus, Charolais, Brahman, and N'Dama. In addition to well-k...

  14. An evolutionary analysis of antigen processing and presentation across different timescales reveals pervasive selection.

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    Diego Forni

    2014-03-01

    Full Text Available The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region. In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones and allowed identification of positive selection events in the human lineage. We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin. Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human

  15. An evolutionary analysis of antigen processing and presentation across different timescales reveals pervasive selection.

    Science.gov (United States)

    Forni, Diego; Cagliani, Rachele; Tresoldi, Claudia; Pozzoli, Uberto; De Gioia, Luca; Filippi, Giulia; Riva, Stefania; Menozzi, Giorgia; Colleoni, Marta; Biasin, Mara; Lo Caputo, Sergio; Mazzotta, Francesco; Comi, Giacomo P; Bresolin, Nereo; Clerici, Mario; Sironi, Manuela

    2014-03-01

    The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage. We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes

  16. Oncotripsy: Targeting cancer cells selectively via resonant harmonic excitation

    CERN Document Server

    Heyden, Stefanie

    2015-01-01

    We investigate a method of selectively targeting cancer cells by means of ultrasound harmonic excitation at their resonance frequency, which we refer to as oncotripsy. The geometric model of the cells takes into account the cytoplasm, nucleus and nucleolus, as well as the plasma membrane and nuclear envelope. Material properties are varied within a pathophysiologically-relevant range. A first modal analysis reveals the existence of a spectral gap between the natural frequencies and, most importantly, resonant growth rates of healthy and cancerous cells. The results of the modal analysis are verified by simulating the fully-nonlinear transient response of healthy and cancerous cells at resonance. The fully nonlinear analysis confirms that cancerous cells can be selectively taken to lysis by the application of carefully tuned ultrasound harmonic excitation while simultaneously leaving healthy cells intact.

  17. Oncotripsy: Targeting cancer cells selectively via resonant harmonic excitation

    Science.gov (United States)

    Heyden, S.; Ortiz, M.

    2016-07-01

    We investigate a method of selectively targeting cancer cells by means of ultrasound harmonic excitation at their resonance frequency, which we refer to as oncotripsy. The geometric model of the cells takes into account the cytoplasm, nucleus and nucleolus, as well as the plasma membrane and nuclear envelope. Material properties are varied within a pathophysiologically-relevant range. A first modal analysis reveals the existence of a spectral gap between the natural frequencies and, most importantly, resonant growth rates of healthy and cancerous cells. The results of the modal analysis are verified by simulating the fully-nonlinear transient response of healthy and cancerous cells at resonance. The fully nonlinear analysis confirms that cancerous cells can be selectively taken to lysis by the application of carefully tuned ultrasound harmonic excitation while simultaneously leaving healthy cells intact.

  18. Sickle Cell: A Selected Resource Bibliography.

    Science.gov (United States)

    National Center for Education in Maternal and Child Health, Washington, DC.

    This annotated, selective bibliography lists the following types of educational and informational material on both sickle cell disease and trait: (1) professional education materials; (2) fact sheets, pamphlets, and brochures; and (3) audiovisual material. A selected list of references is provided for the following topic areas: (1) genetic…

  19. Sensitivity Analysis of Centralized Dynamic Cell Selection

    DEFF Research Database (Denmark)

    Lopez, Victor Fernandez; Alvarez, Beatriz Soret; Pedersen, Klaus I.;

    2016-01-01

    mechanism and solutions involving cell switching in general. Simulation results show that such solutions can greatly benefit from the use of receivers with interference suppression capabilities and a larger number of antennas, with a maximum data rate gain of 120%. High performance gains are observed with...... two different traffic models, and it is not necessary to be able to connect to a large number of cells in order to reap most of the benefits of the centralized dynamic cell selection....

  20. Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes

    OpenAIRE

    Wu, I; Shin, S. C.; Cao, Y; Bender, I K; N Jafari; Feng, G.; Lin, S.; Cidlowski, J. A.; Schleimer, R. P.; Lu, N Z

    2013-01-01

    Induction of T-cell apoptosis contributes to the anti-inflammatory and antineoplastic benefits of glucocorticoids. The glucocorticoid receptor (GR) translational isoforms have distinct proapoptotic activities in osteosarcoma cells. Here we determined whether GR isoforms selectively induce apoptosis in Jurkat T lymphoblastic leukemia cells. Jurkat cells stably expressing individual GR isoforms were generated and treated with vehicle or dexamethasone (DEX). DEX induced apoptosis in cells expres...

  1. Ion-selective microelectrode arrays for cell culture monitoring

    OpenAIRE

    Generelli, Silvia; De Rooij, Nicolas-F.

    2008-01-01

    The design, microfabrication and characterization of a platform comprising an array of ion-selective microelectrodes (µISE) aimed at in vitro cellular physiology and toxicology is described. This study focusses on K+ and Ca2+ monitoring in cell culture environments. A potential promising application of such a platform is based on recent findings in molecular biology, revealing connections between certain diseases, as for example some types of cancer or parkinsonism, and a malfunction in cellu...

  2. Transcriptome sequencing of Eucalyptus camaldulensis seedlings subjected to water stress reveals functional single nucleotide polymorphisms and genes under selection

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    Thumma Bala R

    2012-08-01

    Full Text Available Abstract Background Water stress limits plant survival and production in many parts of the world. Identification of genes and alleles responding to water stress conditions is important in breeding plants better adapted to drought. Currently there are no studies examining the transcriptome wide gene and allelic expression patterns under water stress conditions. We used RNA sequencing (RNA-seq to identify the candidate genes and alleles and to explore the evolutionary signatures of selection. Results We studied the effect of water stress on gene expression in Eucalyptus camaldulensis seedlings derived from three natural populations. We used reference-guided transcriptome mapping to study gene expression. Several genes showed differential expression between control and stress conditions. Gene ontology (GO enrichment tests revealed up-regulation of 140 stress-related gene categories and down-regulation of 35 metabolic and cell wall organisation gene categories. More than 190,000 single nucleotide polymorphisms (SNPs were detected and 2737 of these showed differential allelic expression. Allelic expression of 52% of these variants was correlated with differential gene expression. Signatures of selection patterns were studied by estimating the proportion of nonsynonymous to synonymous substitution rates (Ka/Ks. The average Ka/Ks ratio among the 13,719 genes was 0.39 indicating that most of the genes are under purifying selection. Among the positively selected genes (Ka/Ks > 1.5 apoptosis and cell death categories were enriched. Of the 287 positively selected genes, ninety genes showed differential expression and 27 SNPs from 17 positively selected genes showed differential allelic expression between treatments. Conclusions Correlation of allelic expression of several SNPs with total gene expression indicates that these variants may be the cis-acting variants or in linkage disequilibrium with such variants. Enrichment of apoptosis and cell death gene

  3. Single-Molecule FISH Reveals Non-selective Packaging of Rift Valley Fever Virus Genome Segments.

    Science.gov (United States)

    Wichgers Schreur, Paul J; Kortekaas, Jeroen

    2016-08-01

    The bunyavirus genome comprises a small (S), medium (M), and large (L) RNA segment of negative polarity. Although genome segmentation confers evolutionary advantages by enabling genome reassortment events with related viruses, genome segmentation also complicates genome replication and packaging. Accumulating evidence suggests that genomes of viruses with eight or more genome segments are incorporated into virions by highly selective processes. Remarkably, little is known about the genome packaging process of the tri-segmented bunyaviruses. Here, we evaluated, by single-molecule RNA fluorescence in situ hybridization (FISH), the intracellular spatio-temporal distribution and replication kinetics of the Rift Valley fever virus (RVFV) genome and determined the segment composition of mature virions. The results reveal that the RVFV genome segments start to replicate near the site of infection before spreading and replicating throughout the cytoplasm followed by translocation to the virion assembly site at the Golgi network. Despite the average intracellular S, M and L genome segments approached a 1:1:1 ratio, major differences in genome segment ratios were observed among cells. We also observed a significant amount of cells lacking evidence of M-segment replication. Analysis of two-segmented replicons and four-segmented viruses subsequently confirmed the previous notion that Golgi recruitment is mediated by the Gn glycoprotein. The absence of colocalization of the different segments in the cytoplasm and the successful rescue of a tri-segmented variant with a codon shuffled M-segment suggested that inter-segment interactions are unlikely to drive the copackaging of the different segments into a single virion. The latter was confirmed by direct visualization of RNPs inside mature virions which showed that the majority of virions lack one or more genome segments. Altogether, this study suggests that RVFV genome packaging is a non-selective process. PMID:27548280

  4. Genome-wide Profiling Reveals Remarkable Parallels Between Insertion Site Selection Properties of the MLV Retrovirus and the piggyBac Transposon in Primary Human CD4(+) T Cells.

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    Gogol-Döring, Andreas; Ammar, Ismahen; Gupta, Saumyashree; Bunse, Mario; Miskey, Csaba; Chen, Wei; Uckert, Wolfgang; Schulz, Thomas F; Izsvák, Zsuzsanna; Ivics, Zoltán

    2016-03-01

    The inherent risks associated with vector insertion in gene therapy need to be carefully assessed. We analyzed the genome-wide distributions of Sleeping Beauty (SB) and piggyBac (PB) transposon insertions as well as MLV retrovirus and HIV lentivirus insertions in human CD4(+) T cells with respect to a panel of 40 chromatin states. The distribution of SB transposon insertions displayed the least deviation from random, while the PB transposon and the MLV retrovirus showed unexpected parallels across all chromatin states. Both MLV and PB insertions are enriched at transcriptional start sites (TSSs) and co-localize with BRD4-associated sites. We demonstrate physical interaction between the PB transposase and bromodomain and extraterminal domain proteins (including BRD4), suggesting convergent evolution of a tethering mechanism that directs integrating genetic elements into TSSs. We detect unequal biases across the four systems with respect to targeting genes whose deregulation has been previously linked to serious adverse events in gene therapy clinical trials. The SB transposon has the highest theoretical chance of targeting a safe harbor locus in the human genome. The data underscore the significance of vector choice to reduce the mutagenic load on cells in clinical applications. PMID:26755332

  5. Engineering novel cell surface chemistry for selective tumor cell targeting

    Energy Technology Data Exchange (ETDEWEB)

    Bertozzi, C.R. [Univ. of California, Berkeley, CA (United States)]|[Lawrence Berkeley National Lab., CA (United States)

    1997-12-31

    A common feature of many different cancers is the high expression level of the two monosaccharides sialic acid and fucose within the context of cell-surface associated glycoconjugates. A correlation has been made between hypersialylation and/or hyperfucosylation and the highly metastatic phenotype. Thus, a targeting strategy based on sialic acid or fucose expression would be a powerful tool for the development of new cancer cell-selective therapies and diagnostic agents. We have discovered that ketone groups can be incorporated metabolically into cell-surface associated sialic acids. The ketone is can be covalently ligated with hydrazide functionalized proteins or small molecules under physiological conditions. Thus, we have discovered a mechanism to selectively target hydrazide conjugates to highly sialylated cells such as cancer cells. Applications of this technology to the generation of novel cancer cell-selective toxins and MRI contrast reagents will be discussed, in addition to progress towards the use of cell surface fucose residues as vehicles for ketone expression.

  6. Microgravity-Enhanced Stem Cell Selection

    Science.gov (United States)

    Claudio, Pier Paolo; Valluri, Jagan

    2011-01-01

    Stem cells, both embryonic and adult, promise to revolutionize the practice of medicine in the future. In order to realize this potential, a number of hurdles must be overcome. Most importantly, the signaling mechanisms necessary to control the differentiation of stem cells into tissues of interest remain to be elucidated, and much of the present research on stem cells is focused on this goal. Nevertheless, it will also be essential to achieve large-scale expansion and, in many cases, assemble cells in 3D as transplantable tissues. To this end, microgravity analog bioreactors can play a significant role. Microgravity bioreactors were originally conceived as a tool to study the cellular responses to microgravity. However, the technology can address some of the shortcomings of conventional cell culture systems; namely, the deficiency of mass transport in static culture and high mechanical shear forces in stirred systems. Unexpectedly, the conditions created in the vessel were ideal for 3D cell culture. Recently, investigators have demonstrated the capability of the microgravity bioreactors to expand hematopoietic stem cells compared to static culture, and facilitate the differentiation of umbilical cord stem cells into 3D liver aggregates. Stem cells are capable of differentiating into functional cells. However, there are no reliable methods to induce the stem cells to form specific cells or to gain enough cells for transplantation, which limits their application in clinical therapy. The aim of this study is to select the best experimental setup to reach high proliferation levels by culturing these cells in a microgravity-based bioreactor. In typical cell culture, the cells sediment to the bottom surface of their container and propagate as a one-cell-layer sheet. Prevention of such sedimentation affords the freedom for self-assembly and the propagation of 3D tissue arrays. Suspension of cells is easily achievable using stirred technologies. Unfortunately, in

  7. Recent adaptive events in human brain revealed by meta-analysis of positively selected genes.

    Directory of Open Access Journals (Sweden)

    Yue Huang

    Full Text Available BACKGROUND AND OBJECTIVES: Analysis of positively-selected genes can help us understand how human evolved, especially the evolution of highly developed cognitive functions. However, previous works have reached conflicting conclusions regarding whether human neuronal genes are over-represented among genes under positive selection. METHODS AND RESULTS: We divided positively-selected genes into four groups according to the identification approaches, compiling a comprehensive list from 27 previous studies. We showed that genes that are highly expressed in the central nervous system are enriched in recent positive selection events in human history identified by intra-species genomic scan, especially in brain regions related to cognitive functions. This pattern holds when different datasets, parameters and analysis pipelines were used. Functional category enrichment analysis supported these findings, showing that synapse-related functions are enriched in genes under recent positive selection. In contrast, immune-related functions, for instance, are enriched in genes under ancient positive selection revealed by inter-species coding region comparison. We further demonstrated that most of these patterns still hold even after controlling for genomic characteristics that might bias genome-wide identification of positively-selected genes including gene length, gene density, GC composition, and intensity of negative selection. CONCLUSION: Our rigorous analysis resolved previous conflicting conclusions and revealed recent adaptation of human brain functions.

  8. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    Science.gov (United States)

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  9. Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Iain C. Macaulay

    2016-02-01

    Full Text Available The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment.

  10. Sodium selectivity of Reissner's membrane epithelial cells

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    Kim Kyunghee X

    2011-02-01

    Full Text Available Abstract Background Sodium absorption by Reissner's membrane is thought to contribute to the homeostasis of the volume of cochlear endolymph. It was previously shown that the absorptive transepithelial current was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is the epithelial sodium channel (ENaC, which is composed of the three subunits α-,β- and γ-ENaC. However, other less-selective cation channels have also been observed to be sensitive to benzamil and amiloride. The aim of this study was to determine whether Reissner's membrane epithelial cells could support parasensory K+ absorption via amiloride- and benzamil-sensitive electrogenic pathways. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6196, RT-PCR, and whole-cell patch clamp. Transcript expression analysis of Reissner's membrane detected no amiloride-sensitive acid-sensing ion channels (ASIC1a, ASIC2a, ASIC2b nor amiloride-sensitive cyclic-nucleotide gated channels (CNGA1, CNGA2, CNGA4, CNGB3. By contrast, α-,β- and γ-ENaC were all previously reported as present in Reissner's membrane. The selectivity of the benzamil-sensitive cation currents was observed in whole-cell patch clamp recordings under Cl--free conditions where cations were the only permeant species. The currents were carried by Na+ but not K+, and the permeability of Li+ was greater than that of Na+ in Reissner's membrane. Complete replacement of bath Na+ with the inpermeable cation NMDG+ led to the same inward current as with benzamil in a Na+ bath. Conclusions These results are consistent with the amiloride/benzamil-sensitive absorptive flux of Reissner's membrane mediated by a highly Na+-selective channel that has several key characteristics in common with αβγ-ENaC. The amiloride-sensitive pathway therefore absorbs only Na+ in this epithelium and does not provide a parasensory K+ efflux route from scala

  11. Genomic signatures reveal new evidences for selection of important traits in domestic cattle.

    Science.gov (United States)

    Xu, Lingyang; Bickhart, Derek M; Cole, John B; Schroeder, Steven G; Song, Jiuzhou; Tassell, Curtis P Van; Sonstegard, Tad S; Liu, George E

    2015-03-01

    We investigated diverse genomic selections using high-density single nucleotide polymorphism data of five distinct cattle breeds. Based on allele frequency differences, we detected hundreds of candidate regions under positive selection across Holstein, Angus, Charolais, Brahman, and N'Dama. In addition to well-known genes such as KIT, MC1R, ASIP, GHR, LCORL, NCAPG, WIF1, and ABCA12, we found evidence for a variety of novel and less-known genes under selection in cattle, such as LAP3, SAR1B, LRIG3, FGF5, and NUDCD3. Selective sweeps near LAP3 were then validated by next-generation sequencing. Genome-wide association analysis involving 26,362 Holsteins confirmed that LAP3 and SAR1B were related to milk production traits, suggesting that our candidate regions were likely functional. In addition, haplotype network analyses further revealed distinct selective pressures and evolution patterns across these five cattle breeds. Our results provided a glimpse into diverse genomic selection during cattle domestication, breed formation, and recent genetic improvement. These findings will facilitate genome-assisted breeding to improve animal production and health. PMID:25431480

  12. Frequency Selective Surfaces with Nanoparticles Unit Cell

    Directory of Open Access Journals (Sweden)

    Nga Hung Poon

    2015-09-01

    Full Text Available The frequency selective surface (FSS is a periodic structure with filtering performance for optical and microwave signals. The general periodic arrays made with patterned metallic elements can act as an aperture or patch on a substrate. In this work, two kinds of materials were used to produce unit cells with various patterns. Gold nanoparticles of 25 nm diameter were used to form periodic monolayer arrays by a confined photocatalytic oxidation-based surface modification method. As the other material, silver gel was used to create multiple layers of silver. Due to the ultra-thin nature of the self-assembled gold nanoparticle monolayer, it is very easy to penetrate the FSS with terahertz radiation. However, the isolated silver islands made from silver gel form thicker multiple layers and contribute to much higher reflectance. This work demonstrated that multiple silver layers are more suitable than gold nanoparticles for use in the fabrication of FSS structures.

  13. Selectivity of Direct Methanol Fuel Cell Membranes

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    Antonino S. Aricò

    2015-11-01

    Full Text Available Sulfonic acid-functionalized polymer electrolyte membranes alternative to Nafion® were developed. These were hydrocarbon systems, such as blend sulfonated polyetheretherketone (s-PEEK, new generation perfluorosulfonic acid (PFSA systems, and composite zirconium phosphate–PFSA polymers. The membranes varied in terms of composition, equivalent weight, thickness, and filler and were investigated with regard to their methanol permeation characteristics and proton conductivity for application in direct methanol fuel cells. The behavior of the membrane electrode assemblies (MEA was investigated in fuel cell with the aim to individuate a correlation between membrane characteristics and their performance in a direct methanol fuel cell (DMFC. The power density of the DMFC at 60 °C increased according to a square root-like function of the membrane selectivity. This was defined as the reciprocal of the product between area specific resistance and crossover. The power density achieved at 60 °C for the most promising s-PEEK-based membrane-electrode assembly (MEA was higher than the benchmark Nafion® 115-based MEA (77 mW·cm−2 vs. 64 mW·cm−2. This result was due to a lower methanol crossover (47 mA·cm−2 equivalent current density for s-PEEK vs. 120 mA·cm−2 for Nafion® 115 at 60 °C as recorded at OCV with 2 M methanol and a suitable area specific resistance (0.15 Ohm cm2 for s-PEEK vs. 0.22 Ohm cm2 for Nafion® 115.

  14. Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis

    Science.gov (United States)

    Shen, Keyue; Luk, Samantha; Elman, Jessica; Murray, Ryan; Mukundan, Shilpaa; Parekkadan, Biju

    2016-02-01

    Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

  15. Pathway-specific feedforward circuits between thalamus and neocortex revealed by selective optical stimulation of axons

    OpenAIRE

    Cruikshank, Scott J.; Urabe, Hayato; Nurmikko, Arto V.; Connors, Barry W.

    2010-01-01

    Thalamocortical and corticothalamic pathways mediate bidirectional communication between the thalamus and neocortex. These pathways are entwined, making their study challenging. Here we used lentiviruses to express channelrhodopsin-2 (ChR2), a light-sensitive cation channel, in either thalamocortical or corticothalamic projection cells. Infection occurred only locally but efferent axons and their terminals expressed ChR2 strongly, allowing selective optical activation of each pathway. Laser s...

  16. A clickable neurosteroid photolabel reveals selective Golgi compartmentalization with preferential impact on proximal inhibition.

    Science.gov (United States)

    Jiang, Xiaoping; Shu, Hong-Jin; Krishnan, Kathiresan; Qian, Mingxing; Taylor, Amanda A; Covey, Douglas F; Zorumski, Charles F; Mennerick, Steven

    2016-09-01

    Anesthetic, GABA-active neurosteroids potently augment GABAA receptor function, leading to important behavioral consequences. Neurosteroids and their synthetic analogues are also models for a wide variety of cell-permeant neuroactive compounds. Cell permeation and compartmentalization raise the possibility that these compounds' actions are influenced by their cellular partitioning, but these contributions are not typically considered experimentally or therapeutically. To examine the interplay between cellular accumulation and pharmacodynamics of neurosteroids, we synthesized a novel chemical biology analogue (bio-active, clickable photolabel) of GABA-active neurosteroids. We discovered that the analogue selectively photo-labels neuronal Golgi in rat hippocampal neurons. The active analogue's selective distribution was distinct from endogenous cholesterol and not completely shared by some non-GABA active, neurosteroid-like analogues. On the other hand, the distribution was not enantioselective and did not require energy, in contrast to other recent precedents from the literature. We demonstrate that the soma-selective accumulation can act as a sink or source for steroid actions at plasma-membrane GABA receptors, altering steady-state and time course of effects at somatic GABAA receptors relative to dendritic receptors. Our results suggest a novel mechanism for compartment-selective drug actions at plasma-membrane receptors. PMID:27114255

  17. Structure of a TCR with High Affinity for Self-antigen Reveals Basis for Escape from Negative Selection

    Energy Technology Data Exchange (ETDEWEB)

    Y Yin; Y Li; M Kerzic; R Martin; R Mariuzza

    2011-12-31

    The failure to eliminate self-reactive T cells during negative selection is a prerequisite for autoimmunity. To escape deletion, autoreactive T-cell receptors (TCRs) may form unstable complexes with self-peptide-MHC by adopting suboptimal binding topologies compared with anti-microbial TCRs. Alternatively, escape can occur by weak binding between self-peptides and MHC. We determined the structure of a human autoimmune TCR (MS2-3C8) bound to a self-peptide from myelin basic protein (MBP) and the multiple sclerosis-associated MHC molecule HLA-DR4. MBP is loosely accommodated in the HLA-DR4-binding groove, accounting for its low affinity. Conversely, MS2-3C8 binds MBP-DR4 as tightly as the most avid anti-microbial TCRs. MS2-3C8 engages self-antigen via a docking mode that resembles the optimal topology of anti-foreign TCRs, but is distinct from that of other autoreactive TCRs. Combined with a unique CDR3 conformation, this docking mode compensates for the weak binding of MBP to HLA-DR4 by maximizing interactions between MS2-3C8 and MBP. Thus, the MS2-3C8-MBP-DR4 complex reveals the basis for an alternative strategy whereby autoreactive T cells escape negative selection, yet retain the ability to initiate autoimmunity.

  18. Single molecule imaging reveals differences in microtubule track selection between Kinesin motors.

    Directory of Open Access Journals (Sweden)

    Dawen Cai

    2009-10-01

    Full Text Available Cells generate diverse microtubule populations by polymerization of a common alpha/beta-tubulin building block. How microtubule associated proteins translate microtubule heterogeneity into specific cellular functions is not clear. We evaluated the ability of kinesin motors involved in vesicle transport to read microtubule heterogeneity by using single molecule imaging in live cells. We show that individual Kinesin-1 motors move preferentially on a subset of microtubules in COS cells, identified as the stable microtubules marked by post-translational modifications. In contrast, individual Kinesin-2 (KIF17 and Kinesin-3 (KIF1A motors do not select subsets of microtubules. Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip. Selection of microtubule tracks restricts Kinesin-1 transport of VSVG vesicles to stable microtubules in COS cells whereas KIF17 transport of Kv1.5 vesicles is not restricted to specific microtubules in HL-1 myocytes. These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity. Furthermore, this property enables kinesin motors to segregate membrane trafficking events between stable and dynamic microtubule populations.

  19. Microevolution of nematode miRNAs reveals diverse modes of selection.

    Science.gov (United States)

    Jovelin, Richard; Cutter, Asher D

    2014-01-01

    Micro-RNA (miRNA) genes encode abundant small regulatory RNAs that play key roles during development and in homeostasis by fine tuning and buffering gene expression. This layer of regulatory control over transcriptional networks is preserved by selection across deep evolutionary time, yet selection pressures on individual miRNA genes in contemporary populations remain poorly characterized in any organism. Here, we quantify nucleotide variability for 129 miRNAs in the genome of the nematode Caenorhabditis remanei to understand the microevolution of this important class of regulatory genes. Our analysis of three population samples and C. remanei's sister species revealed ongoing natural selection that constrains evolution of all sequence domains within miRNA hairpins. We also show that new miRNAs evolve faster than older miRNAs but that selection nevertheless favors their persistence. Despite the ongoing importance of purging of new mutations, we discover a trove of >400 natural miRNA sequence variants that include single nucleotide polymorphisms in seed motifs, indels that ablate miRNA functional domains, and origination of new miRNAs by duplication. Moreover, we demonstrate substantial nucleotide divergence of pre-miRNA hairpin alleles between populations and sister species. These findings from the first global survey of miRNA microevolution in Caenorhabditis support the idea that changes in gene expression, mediated through divergence in miRNA regulation, can contribute to phenotypic novelty and adaptation to specific environments in the present day as well as the distant past. PMID:25355809

  20. Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells.

    Science.gov (United States)

    Luo, Yuping; Coskun, Volkan; Liang, Aibing; Yu, Juehua; Cheng, Liming; Ge, Weihong; Shi, Zhanping; Zhang, Kunshan; Li, Chun; Cui, Yaru; Lin, Haijun; Luo, Dandan; Wang, Junbang; Lin, Connie; Dai, Zachary; Zhu, Hongwen; Zhang, Jun; Liu, Jie; Liu, Hailiang; deVellis, Jean; Horvath, Steve; Sun, Yi Eve; Li, Siguang

    2015-05-21

    The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation. PMID:26000486

  1. Cell-to-Cell Diversity in a Synchronized Chlamydomonas Culture As Revealed by Single-Cell Analyses

    OpenAIRE

    Garz, Andreas; Sandmann, Michael; Rading, Michael; Ramm, Sascha; Menzel, Ralf; Steup, Martin

    2012-01-01

    In a synchronized photoautotrophic culture of Chlamydomonas reinhardtii, cell size, cell number, and the averaged starch content were determined throughout the light-dark cycle. For single-cell analyses, the relative cellular starch was quantified by measuring the second harmonic generation (SHG). In destained cells, amylopectin essentially represents the only biophotonic structure. As revealed by various validation procedures, SHG signal intensities are a reliable relative measure of the cel...

  2. Population Variation Reveals Independent Selection toward Small Body Size in Chinese Debao Pony.

    Science.gov (United States)

    Kader, Adiljan; Li, Yan; Dong, Kunzhe; Irwin, David M; Zhao, Qianjun; He, Xiaohong; Liu, Jianfeng; Pu, Yabin; Gorkhali, Neena Amatya; Liu, Xuexue; Jiang, Lin; Li, Xiangchen; Guan, Weijun; Zhang, Yaping; Wu, Dong-Dong; Ma, Yuehui

    2016-01-01

    Body size, one of the most important quantitative traits under evolutionary scrutiny, varies considerably among species and among populations within species. Revealing the genetic basis underlying this variation is very important, particularly in humans where there is a close relationship with diseases and in domestic animals as the selective patterns are associated with improvements in production traits. The Debao pony is a horse breed with small body size that is unique to China; however, it is unknown whether the size-related candidate genes identified in Western breeds also account for the small body size of the Debao pony. Here, we compared individual horses from the Debao population with other two Chinese horse populations using single nucleotide polymorphisms (SNPs) identified with the Equine SNP 65 Bead Chip. The previously reported size-related candidate gene HMGA2 showed a significant signature for selection, consistent with its role observed in human populations. More interestingly, we found a candidate gene TBX3, which had not been observed in previous studies on horse body size that displayed the highest differentiation and most significant association, and thus likely is the dominating factor for the small stature of the Debao pony. Further comparison between the Debao pony and other breeds of horses from around the world demonstrated that TBX3 was selected independently in the Debao pony, suggesting that there were multiple origins of small stature in the horse. PMID:26637467

  3. Mobile eye tracking reveals little evidence for age differences in attentional selection for mood regulation.

    Science.gov (United States)

    Isaacowitz, Derek M; Livingstone, Kimberly M; Harris, Julia A; Marcotte, Stacy L

    2015-04-01

    Two studies are reported representing the first use of mobile eye tracking to study emotion regulation across adulthood. Past research on age differences in attentional deployment using stationary eye tracking has revealed older adults show relatively more positive looking and seem to benefit more moodwise from this looking pattern, compared with younger adults. However, these past studies have greatly constrained the stimuli participants can look at, despite real-world settings providing numerous possibilities for what we choose to look at. The authors therefore used mobile eye tracking to study age differences in attentional selection, as indicated by fixation patterns to stimuli of different valence freely chosen by the participant. In contrast to stationary eye-tracking studies of attentional deployment, Study 1 showed that younger and older individuals generally selected similar proportions of valenced stimuli, and attentional selection had similar effects on mood across age groups. Study 2 replicated this pattern with an adult life span sample including middle-aged individuals. Emotion regulation-relevant attention may thus differ depending on whether stimuli are freely chosen or not. (PsycINFO Database Record PMID:25527965

  4. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

    Directory of Open Access Journals (Sweden)

    Anne L Fletcher

    2011-09-01

    Full Text Available Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  5. Robustness and adaptation reveal plausible cell cycle controlling subnetwork in Saccharomyces cerevisiae.

    Science.gov (United States)

    Huang, Jiun-Yan; Huang, Chi-Wei; Kao, Kuo-Ching; Lai, Pik-Yin

    2013-04-10

    Biological systems are often organized spatially and temporally by multi-scale functional subsystems (modules). A specific subcellular process often corresponds to a subsystem composed of some of these interconnected modules. Accurate identification of system-level modularity organization from the large scale networks can provide valuable information on subsystem models of subcellular processes or physiological phenomena. Computational identification of functional modules from the large scale network is the key approach to solve the complexity of modularity in the past decade, but the overlapping and multi-scale nature of modules often renders unsatisfactory results in these methods. Most current methods for modularity detection are optimization-based and suffered from the drawback of size resolution limit. It is difficult to trace the origin of the unsatisfactory results, which may be due to poor data, inappropriate objective function selection or simply resulted from natural evolution, and hence no system-level accurate modular models for subcellular processes can be offered. Motivated by the idea of evolution with robustness and adaption as guiding principles, we propose a novel approach that can identify significant multi-scale overlapping modules that are sufficiently accurate at the system and subsystem levels, giving biological insights for subcellular processes. The success of our evolution strategy method is demonstrated by applying to the yeast protein-protein interaction network. Functional subsystems of important physiological phenomena can be revealed. In particular, the cell cycle controlling network is selected for detailed discussion. The cell cycle subcellular processes in yeast can be successfully dissected into functional modules of cell cycle control, cell size check point, spindle assembly checkpoint, and DNA damage check point in G2/M and S phases. The interconnections between check points and cell cycle control modules provide clues on the

  6. Dynamics inside the cancer cell attractor reveal cell heterogeneity, limits of stability, and escape.

    Science.gov (United States)

    Li, Qin; Wennborg, Anders; Aurell, Erik; Dekel, Erez; Zou, Jie-Zhi; Xu, Yuting; Huang, Sui; Ernberg, Ingemar

    2016-03-01

    The observed intercellular heterogeneity within a clonal cell population can be mapped as dynamical states clustered around an attractor point in gene expression space, owing to a balance between homeostatic forces and stochastic fluctuations. These dynamics have led to the cancer cell attractor conceptual model, with implications for both carcinogenesis and new therapeutic concepts. Immortalized and malignant EBV-carrying B-cell lines were used to explore this model and characterize the detailed structure of cell attractors. Any subpopulation selected from a population of cells repopulated the whole original basin of attraction within days to weeks. Cells at the basin edges were unstable and prone to apoptosis. Cells continuously changed states within their own attractor, thus driving the repopulation, as shown by fluorescent dye tracing. Perturbations of key regulatory genes induced a jump to a nearby attractor. Using the Fokker-Planck equation, this cell population behavior could be described as two virtual, opposing influences on the cells: one attracting toward the center and the other promoting diffusion in state space (noise). Transcriptome analysis suggests that these forces result from high-dimensional dynamics of the gene regulatory network. We propose that they can be generalized to all cancer cell populations and represent intrinsic behaviors of tumors, offering a previously unidentified characteristic for studying cancer. PMID:26929366

  7. How do I perform hematopoietic progenitor cell selection?

    Science.gov (United States)

    Avecilla, Scott T; Goss, Cheryl; Bleau, Sharon; Tonon, Jo-Ann; Meagher, Richard C

    2016-05-01

    Graft-versus-host disease remains the most important source of morbidity and mortality associated with allogeneic stem cell transplantation. The implementation of hematopoietic progenitor cell (HPC) selection is employed by some stem cell processing facilities to mitigate this complication. Current cell selection methods include reducing the number of unwanted T cells (negative selection) and/or enriching CD34+ hematopoietic stem/progenitors (positive selection) using immunomagnetic beads subjected to magnetic fields within columns to separate out targeted cells. Unwanted side effects of cell selection as a result of T-cell reduction are primary graft failure, increased infection rates, delayed immune reconstitution, possible disease relapse, and posttransplant lymphoproliferative disease. The Miltenyi CliniMACS cell isolation system is the only device currently approved for clinical use by the Food and Drug Administration. It uses magnetic microbeads conjugated with a high-affinity anti-CD34 monoclonal antibody capable of binding to HPCs in marrow, peripheral blood, or umbilical cord blood products. The system results in significantly improved CD34+ cell recoveries (50%-100%) and consistent 3-log CD3+ T-cell reductions compared to previous generations of CD34+ cell selection procedures. In this article, the CliniMACS procedure is described in greater detail and the authors provide useful insight into modifications of the system. Successful implementation of cell selection procedures can have a significant positive clinical effect by greatly increasing the pool of donors for recipients requiring transplants. However, before a program implements cell selection techniques, it is important to consider the time and financial resources required to properly and safely perform these procedures. PMID:26919388

  8. Genome-wide Selective Sweeps in Natural Bacterial Populations Revealed by Time-series Metagenomics

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Leong-Keat; Bendall, Matthew L.; Malfatti, Stephanie; Schwientek, Patrick; Tremblay, Julien; Schackwitz, Wendy; Martin, Joel; Pati, Amrita; Bushnell, Brian; Foster, Brian; Kang, Dongwan; Tringe, Susannah G.; Bertilsson, Stefan; Moran, Mary Ann; Shade, Ashley; Newton, Ryan J.; Stevens, Sarah; McMcahon, Katherine D.; Mamlstrom, Rex R.

    2014-05-12

    Multiple evolutionary models have been proposed to explain the formation of genetically and ecologically distinct bacterial groups. Time-series metagenomics enables direct observation of evolutionary processes in natural populations, and if applied over a sufficiently long time frame, this approach could capture events such as gene-specific or genome-wide selective sweeps. Direct observations of either process could help resolve how distinct groups form in natural microbial assemblages. Here, from a three-year metagenomic study of a freshwater lake, we explore changes in single nucleotide polymorphism (SNP) frequencies and patterns of gene gain and loss in populations of Chlorobiaceae and Methylophilaceae. SNP analyses revealed substantial genetic heterogeneity within these populations, although the degree of heterogeneity varied considerably among closely related, co-occurring Methylophilaceae populations. SNP allele frequencies, as well as the relative abundance of certain genes, changed dramatically over time in each population. Interestingly, SNP diversity was purged at nearly every genome position in one of the Chlorobiaceae populations over the course of three years, while at the same time multiple genes either swept through or were swept from this population. These patterns were consistent with a genome-wide selective sweep, a process predicted by the ecotype model? of diversification, but not previously observed in natural populations.

  9. Genome-wide Selective Sweeps in Natural Bacterial Populations Revealed by Time-series Metagenomics

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Leong-Keat; Bendall, Matthew L.; Malfatti, Stephanie; Schwientek, Patrick; Tremblay, Julien; Schackwitz, Wendy; Martin, Joel; Pati, Amrita; Bushnell, Brian; Foster, Brian; Kang, Dongwan; Tringe, Susannah G.; Bertilsson, Stefan; Moran, Mary Ann; Shade, Ashley; Newton, Ryan J.; Stevens, Sarah; McMahon, Katherine D.; Malmstrom, Rex R.

    2014-06-18

    Multiple evolutionary models have been proposed to explain the formation of genetically and ecologically distinct bacterial groups. Time-series metagenomics enables direct observation of evolutionary processes in natural populations, and if applied over a sufficiently long time frame, this approach could capture events such as gene-specific or genome-wide selective sweeps. Direct observations of either process could help resolve how distinct groups form in natural microbial assemblages. Here, from a three-year metagenomic study of a freshwater lake, we explore changes in single nucleotide polymorphism (SNP) frequencies and patterns of gene gain and loss in populations of Chlorobiaceae and Methylophilaceae. SNP analyses revealed substantial genetic heterogeneity within these populations, although the degree of heterogeneity varied considerably among closely related, co-occurring Methylophilaceae populations. SNP allele frequencies, as well as the relative abundance of certain genes, changed dramatically over time in each population. Interestingly, SNP diversity was purged at nearly every genome position in one of the Chlorobiaceae populations over the course of three years, while at the same time multiple genes either swept through or were swept from this population. These patterns were consistent with a genome-wide selective sweep, a process predicted by the ‘ecotype model’ of diversification, but not previously observed in natural populations.

  10. Miiuy croaker hepcidin gene and comparative analyses reveal evidence for positive selection.

    Directory of Open Access Journals (Sweden)

    Tianjun Xu

    Full Text Available Hepcidin antimicrobial peptide (HAMP is a small cysteine-rich peptide and a key molecule of the innate immune system against bacterial infections. Molecular cloning and genomic characterization of HAMP gene in the miiuy croaker (Miichthys miiuy were reported in this study. The miiuy croaker HAMP was predicted to encode a prepropeptide of 99 amino acids, a tentative RX(K/RR cleavage motif and eight characteristic cysteine residues were also identified. The gene organization is also similar to corresponding genes in mammals and fish consisting of three exons and two introns. Sequence polymorphism analysis showed that only two different sequences were identified and encoded two proteins in six individuals. As reported for most other species, the expression level was highest in liver and an up-regulation of transcription was seen in spleen, intestine and kidney examined at 24 h after injection of pathogenic bacteria, Vibrio anguillarum, the expression pattern implied that miiuy croaker HAMP is an important component of the first line defense against invading pathogens. In addition, we report on the underlying mechanism that maintains sequences diversity among fish and mammalian species, respectively. A series of site-model tests implemented in the CODEML program revealed that moderate positive Darwinian selection is likely to cause the molecular evolution in the fish HAMP2 genes and it also showed that the fish HAMP1 genes and HAMP2 genes under different selection pressures.

  11. Miiuy croaker transferrin gene and evidence for positive selection events reveal different evolutionary patterns.

    Science.gov (United States)

    Sun, Yueyan; Zhu, Zhihuang; Wang, Rixin; Sun, Yuena; Xu, Tianjun

    2012-01-01

    Transferrin (TF) is a protein that plays a central role in iron metabolism. This protein is associated with the innate immune system, which is responsible for disease defense responses after bacterial infection. The clear link between TF and the immune defense mechanism has led researchers to consider TF as a candidate gene for disease resistance. In this study, the Miichthys miiuy (miiuy croaker) TF gene (MIMI-TF) was cloned and characterized. The gene structure consisted of a coding region of 2070 nucleotides divided into 17 exons, as well as a non-coding region that included 16 introns and spans 6757 nucleotides. The deduced MIMI-TF protein consisted of 689 amino acids that comprised a signal peptide and two lobes (N- and C-lobes). MIMI-TF expression was significantly up-regulated after infection with Vibrio anguillarum. A series of model tests implemented in the CODEML program showed that TF underwent a complex evolutionary process. Branch-site models revealed that vertebrate TF was vastly different from that of invertebrates, and that the TF of the ancestors of aquatic and terrestrial organisms underwent different selection pressures. The site models detected 10 positively selected sites in extant TF genes. One site was located in the cleft between the N1 and N2 domains and was expected to affect the capability of TF to bind to or release iron indirectly. In addition, eight sites were found near the TF exterior. Two of these sites, which could have evolved from the competition for iron between pathogenic bacteria and TF, were located in potential pathogen-binding domains. Our results could be used to further investigate the function of TF and the selective mechanisms involved. PMID:22957037

  12. Biomimetic emulsions reveal the effect of homeostatic pressure on cell-cell adhesion

    CERN Document Server

    Pontani, Lea-Laetitia; Viasnoff, Virgile; Brujic, Jasna

    2012-01-01

    Cell-cell contacts in tissues are continuously subject to mechanical forces due to homeostatic pressure and active cytoskeleton dynamics. While much is known about the molecular pathways of adhesion, the role of mechanics is less well understood. To isolate the role of pressure we present a dense packing of functionalized emulsion droplets in which surface interactions are tuned to mimic those of real cells. By visualizing the microstructure in 3D we find that a threshold compression force is necessary to overcome electrostatic repulsion and surface elasticity and establish protein-mediated adhesion. Varying the droplet interaction potential maps out a phase diagram for adhesion as a function of force and salt concentration. Remarkably, fitting the data with our theoretical model predicts binder concentrations in the adhesion areas that are similar to those found in real cells. Moreover, we quantify the adhesion size dependence on the applied force and thus reveal adhesion strengthening with increasing homeos...

  13. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Athanasia D Panopoulos; Margaret Lutz; W Travis Berggren; Kun Zhang; Ronald M Evans; Gary Siuzdak; Juan Carlos Izpisua Belmonte; Oscar Yanes; SergioRuiz; Yasuyuki S Kida; Dinh Diep; Ralf Tautenhahn; Aida Herrerias; Erika M Batchelder; Nongluk Plongthongkum

    2012-01-01

    Metabolism is vital to every aspect of cell function,yet the metabolome of induced pluripotent stem cells (iPSCs)remains largely unexplored.Here we report,using an untargeted metabolomics approach,that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells,and that is characterized by changes in metabolites involved in cellular respiration.Examination of cellular bioenergetics corroborated with our metabolomic analysis,and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency.Interestingly,the bioenergetics of various somatic cells correlated with their reprogramming efficiencies.We further identified metabolites that differ between iPSCs and ESCs,which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming.Our findings are the first to globally analyze the metabolome of iPSCs,and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency,and in evaluating iPSC and ESC equivalence.

  14. Molecular analysis of endothelial progenitor cell (EPC subtypes reveals two distinct cell populations with different identities

    Directory of Open Access Journals (Sweden)

    Simpson David A

    2010-05-01

    Full Text Available Abstract Background The term endothelial progenitor cells (EPCs is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs and outgrowth endothelial cells (OECs. Methods Human blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types. Results Our data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN with links to immunity and inflammation (TLRs, CD14, HLAs, whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs. Conclusions This study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

  15. Aiptasia sp. larvae as a model to reveal mechanisms of symbiont selection in cnidarians

    Science.gov (United States)

    Wolfowicz, Iliona; Baumgarten, Sebastian; Voss, Philipp A.; Hambleton, Elizabeth A.; Voolstra, Christian R.; Hatta, Masayuki; Guse, Annika

    2016-01-01

    Symbiosis, defined as the persistent association between two distinct species, is an evolutionary and ecologically critical phenomenon facilitating survival of both partners in diverse habitats. The biodiversity of coral reef ecosystems depends on a functional symbiosis with photosynthetic dinoflagellates of the highly diverse genus Symbiodinium, which reside in coral host cells and continuously support their nutrition. The mechanisms underlying symbiont selection to establish a stable endosymbiosis in non-symbiotic juvenile corals are unclear. Here we show for the first time that symbiont selection patterns for larvae of two Acropora coral species and the model anemone Aiptasia are similar under controlled conditions. We find that Aiptasia larvae distinguish between compatible and incompatible symbionts during uptake into the gastric cavity and phagocytosis. Using RNA-Seq, we identify a set of candidate genes potentially involved in symbiosis establishment. Together, our data complement existing molecular resources to mechanistically dissect symbiont phagocytosis in cnidarians under controlled conditions, thereby strengthening the role of Aiptasia larvae as a powerful model for cnidarian endosymbiosis establishment. PMID:27582179

  16. Aiptasia sp. larvae as a model to reveal mechanisms of symbiont selection in cnidarians.

    Science.gov (United States)

    Wolfowicz, Iliona; Baumgarten, Sebastian; Voss, Philipp A; Hambleton, Elizabeth A; Voolstra, Christian R; Hatta, Masayuki; Guse, Annika

    2016-01-01

    Symbiosis, defined as the persistent association between two distinct species, is an evolutionary and ecologically critical phenomenon facilitating survival of both partners in diverse habitats. The biodiversity of coral reef ecosystems depends on a functional symbiosis with photosynthetic dinoflagellates of the highly diverse genus Symbiodinium, which reside in coral host cells and continuously support their nutrition. The mechanisms underlying symbiont selection to establish a stable endosymbiosis in non-symbiotic juvenile corals are unclear. Here we show for the first time that symbiont selection patterns for larvae of two Acropora coral species and the model anemone Aiptasia are similar under controlled conditions. We find that Aiptasia larvae distinguish between compatible and incompatible symbionts during uptake into the gastric cavity and phagocytosis. Using RNA-Seq, we identify a set of candidate genes potentially involved in symbiosis establishment. Together, our data complement existing molecular resources to mechanistically dissect symbiont phagocytosis in cnidarians under controlled conditions, thereby strengthening the role of Aiptasia larvae as a powerful model for cnidarian endosymbiosis establishment. PMID:27582179

  17. Ion-Selective Detection with Glass Nanopipette for Living Cells

    Science.gov (United States)

    Takami, T.; Son, J. W.; Kang, E. J.; Deng, X. L.; Kawai, T.; Lee, S.-W.; Park, B. H.

    2013-05-01

    We developed a method to probe local ion concentration with glass nanopipette in which poly(vinyl chloride) membrane containing ionophore for separate ion detection is prepared. Here we demonstrate how ion-selective detections are available for living cells such as HeLa cell, rat vascular myocyte, and neuron cell.

  18. Capillary hydrodynamic chromatography reveals temporal profiles of cell aggregates.

    Science.gov (United States)

    Tang, Ya-Ru; Huang, Hsin-Yi; Hu, Jie-Bi; Rattinam, Rajesh; Li, Chun-Hsien; Chen, Yu-Chie; Urban, Pawel L

    2016-03-01

    Microbial cells are known to form aggregates. Such aggregates can be found in various matrices; for example, functional drinks. Capillary hydrodynamic chromatography (HDC) enables separation of particles by size using nanoliter-scale volumes of samples. Here we propose an approach based on HDC for characterisation of real samples containing aggregated and non-aggregated bacterial and fungal cells. Separation of cells and cell aggregates in HDC arises from the parabolic flow profile under laminar flow conditions. In the presented protocol, hydrodynamic separation is coupled with different on-line and off-line detectors (light absorption/scattering and microscopy). The method has successfully been applied in the monitoring of dynamic changes in the microbiome of probiotic drinks. Chromatographic profiles of yogurt and kefir samples obtained at different times during fermentation are in a good agreement with microscopic images. Moreover, thanks to the implementation of an area imaging detector, capillary HDC could be multiplexed and used to profile spatial gradients in cell suspensions, which arise in the course of sedimentation of cells and cell aggregates. This result shows compatibility of sedimentation analysis and capillary HDC. We believe that the approach may find applications in the profiling of functional foods and other matrices containing aggregated bioparticles. PMID:26873471

  19. Synergistic interaction between selective drugs in cell populations models.

    Directory of Open Access Journals (Sweden)

    Victoria Doldán-Martelli

    Full Text Available The design of selective drugs and combinatorial drug treatments are two of the main focuses in modern pharmacology. In this study we use a mathematical model of chimeric ligand-receptor interaction to show that the combination of selective drugs is synergistic in nature, providing a way to gain optimal selective potential at reduced doses compared to the same drugs when applied individually. We use a cell population model of proliferating cells expressing two different amounts of a target protein to show that both selectivity and synergism are robust against variability and heritability in the cell population. The reduction in the total drug administered due to the synergistic performance of the selective drugs can potentially result in reduced toxicity and off-target interactions, providing a mechanism to improve the treatment of cell-based diseases caused by aberrant gene overexpression, such as cancer and diabetes.

  20. Live cell imaging reveals at novel view of DNA

    International Nuclear Information System (INIS)

    Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) that are the most severe form of DNA damages. Recently, live cell imaging techniques coupled with laser micro-irradiation were used to analyze the spatio-temporal behavior of the NHEJ core factors upon DSB induction in living cells. Based on the live cell imaging studies, we proposed a novel two-phase model for DSB sensing and protein assembly in the NHEJ pathway. This new model provides a novel view of the dynamic protein behavior on DSBs and broad implications for the molecular mechanism of NHEJ. (author)

  1. Transcriptome network analysis reveals candidate genes for renal cell carcinoma

    OpenAIRE

    Wei Zhai; Yun-Fei Xu; Min Liu; Jun-Hua Zheng

    2012-01-01

    Context: Renal cell carcinoma (RCC) is a kidney cancer that originates in renal parenchyma and it is the most common type of kidney cancer with approximately 80% lethal cases. Aims: To interpret the mechanism, explore the regulation of TF-target genes and TF-pathway, and identify the potential key genes of renal cell carcinoma. Settings and Design: After constructing a regulation network from differently expressed genes and transcription factors, pathway regulation network and gene onto...

  2. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    OpenAIRE

    Laura-Roxana Stingaciu; Hugh O’Neill; Michelle Liberton; Urban, Volker S.; Himadri B. Pakrasi; Michael Ohl

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membran...

  3. High Resolution Genomic Scans Reveal Genetic Architecture Controlling Alcohol Preference in Bidirectionally Selected Rat Model.

    Science.gov (United States)

    Lo, Chiao-Ling; Lossie, Amy C; Liang, Tiebing; Liu, Yunlong; Xuei, Xiaoling; Lumeng, Lawrence; Zhou, Feng C; Muir, William M

    2016-08-01

    Investigations on the influence of nature vs. nurture on Alcoholism (Alcohol Use Disorder) in human have yet to provide a clear view on potential genomic etiologies. To address this issue, we sequenced a replicated animal model system bidirectionally-selected for alcohol preference (AP). This model is uniquely suited to map genetic effects with high reproducibility, and resolution. The origin of the rat lines (an 8-way cross) resulted in small haplotype blocks (HB) with a corresponding high level of resolution. We sequenced DNAs from 40 samples (10 per line of each replicate) to determine allele frequencies and HB. We achieved ~46X coverage per line and replicate. Excessive differentiation in the genomic architecture between lines, across replicates, termed signatures of selection (SS), were classified according to gene and region. We identified SS in 930 genes associated with AP. The majority (50%) of the SS were confined to single gene regions, the greatest numbers of which were in promoters (284) and intronic regions (169) with the least in exon's (4), suggesting that differences in AP were primarily due to alterations in regulatory regions. We confirmed previously identified genes and found many new genes associated with AP. Of those newly identified genes, several demonstrated neuronal function involved in synaptic memory and reward behavior, e.g. ion channels (Kcnf1, Kcnn3, Scn5a), excitatory receptors (Grin2a, Gria3, Grip1), neurotransmitters (Pomc), and synapses (Snap29). This study not only reveals the polygenic architecture of AP, but also emphasizes the importance of regulatory elements, consistent with other complex traits. PMID:27490364

  4. Glimpse of natural selection of long-lived T-cell clones in healthy life.

    Science.gov (United States)

    Zhang, Baojun; Jia, Qingzhu; Bock, Cheryl; Chen, Gang; Yu, Haili; Ni, Qingshan; Wan, Ying; Li, Qijing; Zhuang, Yuan

    2016-08-30

    Homeostatic maintenance of T cells with broad clonal diversity is influenced by both continuing output of young T cells from the thymus and ongoing turnover of preexisting clones in the periphery. In the absence of infection, self and commensal antigens are thought to play important roles in selection and homeostatic maintenance of the T-cell pool. Most naïve T cells are short-lived due to lack of antigen encounter, whereas antigen-experienced T cells may survive and persist as long-lived clones. Thus far, little is known about the homeostasis, antigenic specificity, and clonal diversity of long-lived T-cell clones in peripheral lymphoid organs under healthy living conditions. To identify long-lived T-cell clones in mice, we designed a lineage-tracing method to label a wave of T cells produced in the thymus of young mice. After aging the mice for 1.5 y, we found that lineage-tracked T cells consisted of primarily memory-like T cells and T regulatory cells. T-cell receptor repertoire analysis revealed that the lineage-tracked CD4 memory-like T cells and T regulatory cells exhibited age-dependent enrichment of shared clonotypes. Furthermore, these shared clonotypes were found across different mice maintained in the same housing condition. These findings suggest that nonrandom and shared antigens are involved in controlling selection, retention, and immune tolerance of long-lived T-cell clones under healthy living conditions. PMID:27535935

  5. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. T Cell Adolescence: Maturation Events Beyond Positive Selection.

    Science.gov (United States)

    Hogquist, Kristin A; Xing, Yan; Hsu, Fan-Chi; Shapiro, Virginia Smith

    2015-08-15

    Single-positive thymocytes that successfully complete positive and negative selection must still undergo one final step, generally termed T cell maturation, before they gain functional competency and enter the long-lived T cell pool. Maturation initiates after positive selection in single-positive thymocytes and continues in the periphery in recent thymic emigrants, before these newly produced T cells gain functional competency and are ready to participate in the immune response as peripheral naive T cells. Recent work using genetically altered mice demonstrates that T cell maturation is not a single process, but a series of steps that occur independently and sequentially after positive selection. This review focuses on the changes that occur during T cell maturation, as well as the molecules and pathways that are critical at each step. PMID:26254267

  7. Advancing tandem solar cells by spectrally selective multilayer intermediate reflectors.

    Science.gov (United States)

    Hoffmann, Andre; Paetzold, Ulrich W; Zhang, Chao; Merdzhanova, Tsvetelina; Lambertz, Andreas; Ulbrich, Carolin; Bittkau, Karsten; Rau, Uwe

    2014-08-25

    Thin-film silicon tandem solar cells are composed of an amorphous silicon top cell and a microcrystalline silicon bottom cell, stacked and connected in series. In order to match the photocurrents of the top cell and the bottom cell, a proper photon management is required. Up to date, single-layer intermediate reflectors of limited spectral selectivity are applied to match the photocurrents of the top and the bottom cell. In this paper, we design and prototype multilayer intermediate reflectors based on aluminum doped zinc oxide and doped microcrystalline silicon oxide with a spectrally selective reflectance allowing for improved current matching and an overall increase of the charge carrier generation. The intermediate reflectors are successfully integrated into state-of-the-art tandem solar cells resulting in an increase of overall short-circuit current density by 0.7 mA/cm(2) in comparison to a tandem solar cell with the standard single-layer intermediate reflector. PMID:25322181

  8. The Slow:Fast substitution ratio reveals changing patterns of natural selection in gamma-proteobacterial genomes

    Energy Technology Data Exchange (ETDEWEB)

    Alm, Eric; Shapiro, B. Jesse

    2009-04-15

    Different microbial species are thought to occupy distinct ecological niches, subjecting each species to unique selective constraints, which may leave a recognizable signal in their genomes. Thus, it may be possible to extract insight into the genetic basis of ecological differences among lineages by identifying unusual patterns of substitutions in orthologous gene or protein sequences. We use the ratio of substitutions in slow versus fast-evolving sites (nucleotides in DNA, or amino acids in protein sequence) to quantify deviations from the typical pattern of selective constraint observed across bacterial lineages. We propose that elevated S:F in one branch (an excess of slow-site substitutions) can indicate a functionally-relevant change, due to either positive selection or relaxed evolutionary constraint. In a genome-wide comparative study of gamma-proteobacterial proteins, we find that cell-surface proteins involved with motility and secretion functions often have high S:F ratios, while information-processing genes do not. Change in evolutionary constraints in some species is evidenced by increased S:F ratios within functionally-related sets of genes (e.g., energy production in Pseudomonas fluorescens), while other species apparently evolve mostly by drift (e.g., uniformly elevated S:F across most genes in Buchnera spp.). Overall, S:F reveals several species-specific, protein-level changes with potential functional/ecological importance. As microbial genome projects yield more species-rich gene-trees, the S:F ratio will become an increasingly powerful tool for uncovering functional genetic differences among species.

  9. Selectable-Tip Corrosion-Testing Electrochemical Cell

    Science.gov (United States)

    Lomness, Janice; Hintze, Paul

    2008-01-01

    The figure depicts aspects of an electrochemical cell for pitting- corrosion tests of material specimens. The cell is designed to generate a region of corrosion having a pit diameter determined by the diameter of a selectable tip. The average depth of corrosion is controlled by controlling the total electric charge passing through the cell in a test. The cell is also designed to produce minimal artifacts associated with crevice corrosion. There are three selectable tips, having diameters of 0.1 in. (0.254 cm), 0.3 in. (0.762 cm), and 0.6 in. (1.524 cm), respectively.

  10. T cell depleted haploidentical transplantation: positive selection

    Directory of Open Access Journals (Sweden)

    Franco Aversa

    2011-06-01

    Full Text Available Interest in mismatched transplantation arises from the fact that a suitable one-haplotype mismatched donor is immediately available for virtually all patients, particularly for those who urgently need an allogenic transplant. Work on one haplotype-mismatched transplants has been proceeding for over 20 years all over the world and novel transplant techniques have been developed. Some centres have focused on the conditioning regimens and post transplant immune suppression; others have concentrated on manipulating the graft which may be a megadose of extensively T celldepleted or unmanipulated progenitor cells. Excellent engraftment rates are associated with a very low incidence of acute and chronic GVHD and regimen-related mortality even in patients who are over 50 years old. Overall, event-free survival and transplant-related mortality compare favourably with reports on transplants from sources of stem cells other than the matched sibling.

  11. Nanoparticle-Based Immunocytochemistry Reveals Microarchitecture of the Cell Nucleus

    Czech Academy of Sciences Publication Activity Database

    Hozák, Pavel

    Kyoto : International Federation of Societies for Histochemistry and Cytochemistry, Japan Society of Histochemistry, 2012. [14th International Congress of Histochemistry and Cytochemistry. Kyoto (JP), 26.08.2012-29.08.2012] R&D Projects: GA ČR GAP305/11/2232; GA MŠk LC545; GA MŠk(CZ) LC06063; GA MPO(CZ) FRTI3588 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : PIP2 * NMI * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology

  12. Nanoparticle-based immunocytochemistry reveals microarchitecture of the cell nucleus

    Czech Academy of Sciences Publication Activity Database

    Hozák, Pavel

    Mérida : CIASEM, AMM, 2011. ---. [Inter-American Congress on Microscopy /11./. 25.09.2011-29.09.2011, Mérida] R&D Projects: GA ČR GAP305/11/2232; GA ČR(CZ) GD204/09/H084; GA MŠk LC545; GA MŠk(CZ) LC06063; GA AV ČR KAN200520704 Institutional research plan: CEZ:AV0Z50520514 Keywords : nanoparticles * immunogold detection * histochemistry * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology

  13. Nanoparticle-based immunocytochemistry reveals microarchitecture of the cell nucleus

    Czech Academy of Sciences Publication Activity Database

    Hozák, Pavel

    Istanbul: Turkish Society for Electron Microscopy, 2011. ---. [National Electron Microscopy Congress /20./. 25.10.2011-28.10.2011, Kemer] R&D Projects: GA ČR GAP305/11/2232; GA ČR(CZ) GD204/09/H084; GA MŠk LC545; GA MŠk(CZ) LC06063; GA MŠk 2B06063; GA AV ČR KAN200520704 Institutional research plan: CEZ:AV0Z50520514 Keywords : nanoparticles * immunogold detection * histochemistry * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology

  14. Punishment-Induced Behavioral and Neurophysiological Variability Reveals Dopamine-Dependent Selection of Kinematic Movement Parameters

    OpenAIRE

    Galea, J. M.; Ruge, D.; Buijink, A.; Bestmann, S.; Rothwell, J. C.

    2013-01-01

    Action selection describes the high-level process which selects between competing movements. In animals, behavioural variability is critical for the motor exploration required to select the action which optimizes reward and minimizes cost/punishment, and is guided by dopamine (DA). The aim of this study was to test in humans whether low-level movement parameters are affected by punishment and reward in ways similar to high-level action selection. Moreover, we addressed the proposed dependence...

  15. Single cell activity reveals direct electron transfer in methanotrophic consortia

    Science.gov (United States)

    McGlynn, Shawn E.; Chadwick, Grayson L.; Kempes, Christopher P.; Orphan, Victoria J.

    2015-10-01

    Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer.

  16. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    Science.gov (United States)

    Stingaciu, Laura-Roxana; O'Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.

  17. Natural variation of heterokaryon incompatibility gene het-c in Podospora anserina reveals diversifying selection.

    Science.gov (United States)

    Bastiaans, Eric; Debets, Alfons J M; Aanen, Duur K; van Diepeningen, Anne D; Saupe, Sven J; Paoletti, Mathieu

    2014-04-01

    In filamentous fungi, allorecognition takes the form of heterokaryon incompatibility, a cell death reaction triggered when genetically distinct hyphae fuse. Heterokaryon incompatibility is controlled by specific loci termed het-loci. In this article, we analyzed the natural variation in one such fungal allorecognition determinant, the het-c heterokaryon incompatibility locus of the filamentous ascomycete Podospora anserina. The het-c locus determines an allogenic incompatibility reaction together with two unlinked loci termed het-d and het-e. Each het-c allele is incompatible with a specific subset of the het-d and het-e alleles. We analyzed variability at the het-c locus in a population of 110 individuals, and in additional isolates from various localities. We identified a total of 11 het-c alleles, which define 7 distinct incompatibility specificity classes in combination with the known het-d and het-e alleles. We found that the het-c allorecognition gene of P. anserina is under diversifying selection. We find a highly unequal allele distribution of het-c in the population, which contrasts with the more balanced distribution of functional groups of het-c based on their allorecognition function. One explanation for the observed het-c diversity in the population is its function in allorecognition. However, alleles that are most efficient in allorecognition are rare. An alternative and not exclusive explanation for the observed diversity is that het-c is involved in pathogen recognition. In Arabidopsis thaliana, a homolog of het-c is a pathogen effector target, supporting this hypothesis. We hypothesize that the het-c diversity in P. anserina results from both its functions in pathogen-defense, and allorecognition. PMID:24448643

  18. Naturally death-resistant precursor cells revealed as the origin of retinoblastoma

    DEFF Research Database (Denmark)

    Trinh, Emmanuelle; Lazzerini Denchi, Eros; Helin, Kristian

    2004-01-01

    The molecular mechanisms and the cell-of-origin leading to retinoblastoma are not well defined. In this issue of Cancer Cell, Bremner and colleagues describe the first inheritable model of retinoblastoma, revealing that loss of the pocket proteins pRb and p107 deregulates cell cycle exit in retin...

  19. Transcriptional activity around bacterial cell death reveals molecular biomarkers for cell viability

    Directory of Open Access Journals (Sweden)

    Schuren Frank H

    2008-12-01

    Full Text Available Abstract Background In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis. Results We present microbiological, cytological and molecular analyses of the physiological response to lethal heat stress under accurately defined conditions through systematic sampling of bacteria from a single culture exposed to gradually increasing temperatures. We identified a coherent transcriptional program including known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase. Conclusion The observed coordinated gene expression continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce. Transcription of a very limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria – but silent in dead bacteria-include those of essential genes encoding chaperones of the protein folding machinery and can serve as molecular biomarkers for bacterial cell viability.

  20. A nanocomplex that is both tumor cell-selective and cancer gene-specific for anaplastic large cell lymphoma

    Directory of Open Access Journals (Sweden)

    Zu Youli

    2011-01-01

    Full Text Available Abstract Background Many in vitro studies have demonstrated that silencing of cancerous genes by siRNAs is a potential therapeutic approach for blocking tumor growth. However, siRNAs are not cell type-selective, cannot specifically target tumor cells, and therefore have limited in vivo application for siRNA-mediated gene therapy. Results In this study, we tested a functional RNA nanocomplex which exclusively targets and affects human anaplastic large cell lymphoma (ALCL by taking advantage of the abnormal expression of CD30, a unique surface biomarker, and the anaplastic lymphoma kinase (ALK gene in lymphoma cells. The nanocomplexes were formulated by incorporating both ALK siRNA and a RNA-based CD30 aptamer probe onto nano-sized polyethyleneimine-citrate carriers. To minimize potential cytotoxicity, the individual components of the nanocomplexes were used at sub-cytotoxic concentrations. Dynamic light scattering showed that formed nanocomplexes were ~140 nm in diameter and remained stable for more than 24 hours in culture medium. Cell binding assays revealed that CD30 aptamer probes selectively targeted nanocomplexes to ALCL cells, and confocal fluorescence microscopy confirmed intracellular delivery of the nanocomplex. Cell transfection analysis showed that nanocomplexes silenced genes in an ALCL cell type-selective fashion. Moreover, exposure of ALCL cells to nanocomplexes carrying both ALK siRNAs and CD30 RNA aptamers specifically silenced ALK gene expression, leading to growth arrest and apoptosis. Conclusions Taken together, our findings indicate that this functional RNA nanocomplex is both tumor cell type-selective and cancer gene-specific for ALCL cells.

  1. Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation

    OpenAIRE

    Sugiyama, Takuya; Benitez, Cecil M.; Ghodasara, Amar; Liu, Lucy; McLean, Graeme W.; Lee, Jonghyeob; Blauwkamp, Timothy A; Nusse, Roeland; Wright, Christopher V.E.; Gu, Guoqiang; Kim, Seung K.

    2013-01-01

    Developmental biology is challenged to reveal the function of numerous candidate genes implicated by recent genome-scale studies as regulators of organ development and diseases. Recapitulating organogenesis from purified progenitor cells that can be genetically manipulated would provide powerful opportunities to dissect such gene functions. Here we describe systems for reconstructing pancreas development, including islet β-cell and α-cell differentiation, from single fetal progenitor cells. A...

  2. Induction and Selection of Sox17-Expressing Endoderm Cells Generated from Murine Embryonic Stem Cells

    OpenAIRE

    Schroeder, I.; Sulzbacher, S.; T. Nolden; Fuchs, J.; Czarnota, J.; Meisterfeld, R.; Himmelbauer, H.; Wobus, A

    2014-01-01

    Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transc...

  3. Conditional IFNAR1 ablation reveals distinct requirements of Type I IFN signaling for NK cell maturation and tumor surveillance

    Science.gov (United States)

    Mizutani, Tatsuaki; Neugebauer, Nina; Putz, Eva M.; Moritz, Nadine; Simma, Olivia; Zebedin-Brandl, Eva; Gotthardt, Dagmar; Warsch, Wolfgang; Eckelhart, Eva; Kantner, Hans-Peter; Kalinke, Ulrich; Lienenklaus, Stefan; Weiss, Siegfried; Strobl, Birgit; Müller, Mathias; Sexl, Veronika; Stoiber, Dagmar

    2012-01-01

    Mice with an impaired Type I interferon (IFN) signaling (IFNAR1- and IFNβ-deficient mice) display an increased susceptibility toward v-ABL-induced B-cell leukemia/lymphoma. The enhanced leukemogenesis in the absence of an intact Type I IFN signaling is caused by alterations within the tumor environment. Deletion of Ifnar1 in tumor cells (as obtained in Ifnar1f/f CD19-Cre mice) failed to impact on disease latency or type. In line with this observation, the initial transformation and proliferative capacity of tumor cells were unaltered irrespective of whether the cells expressed IFNAR1 or not. v-ABL-induced leukemogenesis is mainly subjected to natural killer (NK) cell-mediated tumor surveillance. Thus, we concentrated on NK cell functions in IFNAR1 deficient animals. Ifnar1-/- NK cells displayed maturation defects as well as an impaired cytolytic activity. When we deleted Ifnar1 selectively in mature NK cells (by crossing Ncr1-iCre mice to Ifnar1f/f animals), maturation was not altered. However, NK cells derived from Ifnar1f/f Ncr1-iCre mice showed a significant cytolytic defect in vitro against the hematopoietic cell lines YAC-1 and RMA-S, but not against the melanoma cell line B16F10. Interestingly, this defect was not related to an in vivo phenotype as v-ABL-induced leukemogenesis was unaltered in Ifnar1f/f Ncr1-iCre compared with Ifnar1f/f control mice. Moreover, the ability of Ifnar1f/f Ncr1-iCre NK cells to kill B16F10 melanoma cells was unaltered, both in vitro and in vivo. Our data reveal that despite the necessity for Type I IFN in NK cell maturation the expression of IFNAR1 on mature murine NK cells is not required for efficient tumor surveillance. PMID:23170251

  4. Perceptual load manipulation reveals sensitivity of the face-selective N170 to attention.

    Science.gov (United States)

    Mohamed, Tarik N; Neumann, Markus F; Schweinberger, Stefan R

    2009-05-27

    It has been controversial whether the face-sensitive N170 is affected by selective attention. We manipulated attention sensu Lavie's perceptual load theory to short (200 ms) presentations of task-irrelevant unfamiliar faces or houses, while participants identified superimposed target letters 'X' versus 'N'. These targets were strings of either six identical (low load) or six different letters (high load). Under low load, we found a prominent face-selective N170 response. Under high load, however, we not only observed a dramatic reduction of the face N170 but also an unexpected enhancement of the house N170, such that face selectivity was almost completely lost. We conclude that the early stages of face processing indexed by the N170 strongly depend on selective attention. PMID:19369907

  5. Genome-wide analysis reveals selection for important traits in domestic horse breeds.

    Science.gov (United States)

    Petersen, Jessica L; Mickelson, James R; Rendahl, Aaron K; Valberg, Stephanie J; Andersson, Lisa S; Axelsson, Jeanette; Bailey, Ernie; Bannasch, Danika; Binns, Matthew M; Borges, Alexandre S; Brama, Pieter; da Câmara Machado, Artur; Capomaccio, Stefano; Cappelli, Katia; Cothran, E Gus; Distl, Ottmar; Fox-Clipsham, Laura; Graves, Kathryn T; Guérin, Gérard; Haase, Bianca; Hasegawa, Telhisa; Hemmann, Karin; Hill, Emmeline W; Leeb, Tosso; Lindgren, Gabriella; Lohi, Hannes; Lopes, Maria Susana; McGivney, Beatrice A; Mikko, Sofia; Orr, Nicholas; Penedo, M Cecilia T; Piercy, Richard J; Raekallio, Marja; Rieder, Stefan; Røed, Knut H; Swinburne, June; Tozaki, Teruaki; Vaudin, Mark; Wade, Claire M; McCue, Molly E

    2013-01-01

    Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an F(ST)-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse. PMID:23349635

  6. What do spring migrants reveal about sex and host selection in the melon aphid?

    OpenAIRE

    Thomas Sophie; Boissot Nathalie; Vanlerberghe-Masutti Flavie

    2012-01-01

    Abstract Background Host plants exert considerable selective pressure on aphids because the plants constitute their feeding, mating and oviposition sites. Therefore, host specialisation in aphids evolves through selection of the behavioural and chemical mechanisms of host-plant location and recognition, and through metabolic adaptation to the phloem content of the host plant. How these adaptive traits evolve in an aphid species depends on the complexity of the annual life cycle of that specie...

  7. Experimental selection reveals a trade-off between fecundity and lifespan in the coliphage Qß

    OpenAIRE

    García-Villada, Libertad; Drake, John W.

    2013-01-01

    Understanding virus evolution is key for improving ways to counteract virus-borne diseases. Results from comparative analyses have previously suggested a trade-off between fecundity and lifespan for viruses that infect the bacterium Escherichia coli (i.e. for coliphages), which, if confirmed, would define a particular constraint on the evolution of virus fecundity. Here, the occurrence of such a trade-off is investigated through a selection experiment using the coliphage Qß. Selection was app...

  8. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    Science.gov (United States)

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

    1992-09-01

    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  9. Proteomic analysis of imatinib-resistant CML-T1 cells reveals calcium homeostasis as a potential therapeutic target

    Science.gov (United States)

    Toman, O.; Kabickova, T.; Vit, O.; Fiser, R.; Polakova, K. Machova; Zach, J.; Linhartova, J.; Vyoral, D.; Petrak, J.

    2016-01-01

    Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib-resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients. PMID:27430982

  10. Selective Cell Targeting with Light-Absorbing Microparticles and Nanoparticles

    OpenAIRE

    Pitsillides, Costas M; Joe, Edwin K.; Wei, Xunbin; Anderson, R. Rox; Lin, Charles P.

    2003-01-01

    We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond ...

  11. Directional summation in non-direction selective retinal ganglion cells.

    Directory of Open Access Journals (Sweden)

    Syed Y Abbas

    Full Text Available Retinal ganglion cells receive inputs from multiple bipolar cells which must be integrated before a decision to fire is made. Theoretical studies have provided clues about how this integration is accomplished but have not directly determined the rules regulating summation of closely timed inputs along single or multiple dendrites. Here we have examined dendritic summation of multiple inputs along On ganglion cell dendrites in whole mount rat retina. We activated inputs at targeted locations by uncaging glutamate sequentially to generate apparent motion along On ganglion cell dendrites in whole mount retina. Summation was directional and dependent13 on input sequence. Input moving away from the soma (centrifugal resulted in supralinear summation, while activation sequences moving toward the soma (centripetal were linear. Enhanced summation for centrifugal activation was robust as it was also observed in cultured retinal ganglion cells. This directional summation was dependent on hyperpolarization activated cyclic nucleotide-gated (HCN channels as blockade with ZD7288 eliminated directionality. A computational model confirms that activation of HCN channels can override a preference for centripetal summation expected from cell anatomy. This type of direction selectivity could play a role in coding movement similar to the axial selectivity seen in locust ganglion cells which detect looming stimuli. More generally, these results suggest that non-directional retinal ganglion cells can discriminate between input sequences independent of the retina network.

  12. Directional summation in non-direction selective retinal ganglion cells.

    Science.gov (United States)

    Abbas, Syed Y; Hamade, Khaldoun C; Yang, Ellen J; Nawy, Scott; Smith, Robert G; Pettit, Diana L

    2013-01-01

    Retinal ganglion cells receive inputs from multiple bipolar cells which must be integrated before a decision to fire is made. Theoretical studies have provided clues about how this integration is accomplished but have not directly determined the rules regulating summation of closely timed inputs along single or multiple dendrites. Here we have examined dendritic summation of multiple inputs along On ganglion cell dendrites in whole mount rat retina. We activated inputs at targeted locations by uncaging glutamate sequentially to generate apparent motion along On ganglion cell dendrites in whole mount retina. Summation was directional and dependent13 on input sequence. Input moving away from the soma (centrifugal) resulted in supralinear summation, while activation sequences moving toward the soma (centripetal) were linear. Enhanced summation for centrifugal activation was robust as it was also observed in cultured retinal ganglion cells. This directional summation was dependent on hyperpolarization activated cyclic nucleotide-gated (HCN) channels as blockade with ZD7288 eliminated directionality. A computational model confirms that activation of HCN channels can override a preference for centripetal summation expected from cell anatomy. This type of direction selectivity could play a role in coding movement similar to the axial selectivity seen in locust ganglion cells which detect looming stimuli. More generally, these results suggest that non-directional retinal ganglion cells can discriminate between input sequences independent of the retina network. PMID:23516351

  13. Analysis of Adaptive Evolution in Lyssavirus Genomes Reveals Pervasive Diversifying Selection during Species Diversification

    Directory of Open Access Journals (Sweden)

    Carolina M. Voloch

    2014-11-01

    Full Text Available Lyssavirus is a diverse genus of viruses that infect a variety of mammalian hosts, typically causing encephalitis. The evolution of this lineage, particularly the rabies virus, has been a focus of research because of the extensive occurrence of cross-species transmission, and the distinctive geographical patterns present throughout the diversification of these viruses. Although numerous studies have examined pattern-related questions concerning Lyssavirus evolution, analyses of the evolutionary processes acting on Lyssavirus diversification are scarce. To clarify the relevance of positive natural selection in Lyssavirus diversification, we conducted a comprehensive scan for episodic diversifying selection across all lineages and codon sites of the five coding regions in lyssavirus genomes. Although the genomes of these viruses are generally conserved, the glycoprotein (G, RNA-dependent RNA polymerase (L and polymerase (P genes were frequently targets of adaptive evolution during the diversification of the genus. Adaptive evolution is particularly manifest in the glycoprotein gene, which was inferred to have experienced the highest density of positively selected codon sites along branches. Substitutions in the L gene were found to be associated with the early diversification of phylogroups. A comparison between the number of positively selected sites inferred along the branches of RABV population branches and Lyssavirus intespecies branches suggested that the occurrence of positive selection was similar on the five coding regions of the genome in both groups.

  14. Artificial Selection Reveals High Genetic Variation in Phenology at the Trailing Edge of a Species Range.

    Science.gov (United States)

    Sheth, Seema Nayan; Angert, Amy Lauren

    2016-02-01

    Species responses to climate change depend on the interplay of migration and adaptation, yet we know relatively little about the potential for adaptation. Genetic adaptations to climate change often involve shifts in the timing of phenological events, such as flowering. If populations at the edge of a species range have lower genetic variation in phenological traits than central populations, then their persistence under climate change could be threatened. To test this hypothesis, we performed artificial selection experiments using the scarlet monkeyflower (Mimulus cardinalis) and compared genetic variation in flowering time among populations at the latitudinal center, northern edge, and southern edge of the species range. We also assessed whether selection on flowering time yielded correlated responses in functional traits, potentially representing a cost associated with early or late flowering. Contrary to prediction, southern populations exhibited greater responses to selection on flowering time than central or northern populations. Further, selection for early flowering resulted in correlated increases in specific leaf area and leaf nitrogen, whereas selection for late flowering led to decreases in these traits. These results provide critical insights about how spatial variation in the potential for adaptation may affect population persistence under changing climates. PMID:26807746

  15. Meaning Making: What Reflective Essays Reveal about Biology Students' Conceptions about Natural Selection

    Science.gov (United States)

    Balgopal, Meena M.; Montplaisir, Lisa M.

    2011-01-01

    The process of reflective writing can play a central role in making meaning as learners process new information and connect it to prior knowledge. An examination of the written discourse can therefore be revealing of learners' cognitive understanding and affective (beliefs, feelings, motivation to learn) responses to concepts. Despite reflective…

  16. Analysis of neural subtypes reveals selective mitochondrial dysfunction in dopaminergic neurons from parkin mutants

    OpenAIRE

    Jonathon L Burman; Yu, Selina; Angela C. Poole; Decal, Richard B.; Pallanck, Leo

    2012-01-01

    Studies of the familial Parkinson disease-related proteins PINK1 and Parkin have demonstrated that these factors promote the fragmentation and turnover of mitochondria following treatment of cultured cells with mitochondrial depolarizing agents. Whether PINK1 or Parkin influence mitochondrial quality control under normal physiological conditions in dopaminergic neurons, a principal cell type that degenerates in Parkinson disease, remains unclear. To address this matter, we developed a method ...

  17. Physics of sinking and selection of plankton cell size

    Energy Technology Data Exchange (ETDEWEB)

    Sciascia, R., E-mail: r.sciascia@isac.cnr.it [Institute of Atmospheric Sciences and Climate, CNR, Corso Fiume, 4, 10133 Torino (Italy); Doctorate Program in Fluid Dynamics, Politecnico di Torino (Italy); De Monte, S. [CNRS, UMR 7625 “Ecologie et Evolution”, Paris, F-75005 (France); Université Pierre et Marie Curie-Paris 6, UMR 7625 “Ecologie et Evolution”, Paris, F-75005 (France); Institut de Biologie de l' Ecole Normale Supérieure, UMR 7625 “Ecologie et Evolution”, Paris, F-75005 (France); Provenzale, A. [Institute of Atmospheric Sciences and Climate, CNR, Corso Fiume, 4, 10133 Torino (Italy)

    2013-02-04

    Gravitational sinking in the water column is known to affect size composition of planktonic communities. One important driver toward the reduction of plankton size is the fact that larger cells tend to sink faster below the euphotic layer. In this work, we discuss the role of gravitational sinking in driving cell size selection, showing that the outcome of phytoplankton competition is determined by the dependence of sinking velocity on cell size, shape, and on the temporal variability associated with turbulence. This opens a question on whether regional modulations of the turbulence intensity could affect size distribution of planktonic communities.

  18. Genome-wide view of genetic diversity reveals paths of selection and cultivar differentiation in peach domestication.

    Science.gov (United States)

    Akagi, Takashi; Hanada, Toshio; Yaegaki, Hideaki; Gradziel, Thomas M; Tao, Ryutaro

    2016-06-01

    Domestication and cultivar differentiation are requisite processes for establishing cultivated crops. These processes inherently involve substantial changes in population structure, including those from artificial selection of key genes. In this study, accessions of peach (Prunus persica) and its wild relatives were analysed genome-wide to identify changes in genetic structures and gene selections associated with their differentiation. Analysis of genome-wide informative single-nucleotide polymorphism loci revealed distinct changes in genetic structures and delineations among domesticated peach and its wild relatives and among peach landraces and modern fruit (F) and modern ornamental (O-A) cultivars. Indications of distinct changes in linkage disequilibrium extension/decay and of strong population bottlenecks or inbreeding were identified. Site frequency spectrum- and extended haplotype homozygosity-based evaluation of genome-wide genetic diversities supported selective sweeps distinguishing the domesticated peach from its wild relatives and each F/O-A cluster from the landrace clusters. The regions with strong selective sweeps harboured promising candidates for genes subjected to selection. Further sequence-based evaluation further defined the candidates and revealed their characteristics. All results suggest opportunities for identifying critical genes associated with each differentiation by analysing genome-wide genetic diversity in currently established populations. This approach obviates the special development of genetic populations, which is particularly difficult for long-lived tree crops. PMID:27085183

  19. Genome-wide view of genetic diversity reveals paths of selection and cultivar differentiation in peach domestication

    Science.gov (United States)

    Akagi, Takashi; Hanada, Toshio; Yaegaki, Hideaki; Gradziel, Thomas M.; Tao, Ryutaro

    2016-01-01

    Domestication and cultivar differentiation are requisite processes for establishing cultivated crops. These processes inherently involve substantial changes in population structure, including those from artificial selection of key genes. In this study, accessions of peach (Prunus persica) and its wild relatives were analysed genome-wide to identify changes in genetic structures and gene selections associated with their differentiation. Analysis of genome-wide informative single-nucleotide polymorphism loci revealed distinct changes in genetic structures and delineations among domesticated peach and its wild relatives and among peach landraces and modern fruit (F) and modern ornamental (O-A) cultivars. Indications of distinct changes in linkage disequilibrium extension/decay and of strong population bottlenecks or inbreeding were identified. Site frequency spectrum- and extended haplotype homozygosity-based evaluation of genome-wide genetic diversities supported selective sweeps distinguishing the domesticated peach from its wild relatives and each F/O-A cluster from the landrace clusters. The regions with strong selective sweeps harboured promising candidates for genes subjected to selection. Further sequence-based evaluation further defined the candidates and revealed their characteristics. All results suggest opportunities for identifying critical genes associated with each differentiation by analysing genome-wide genetic diversity in currently established populations. This approach obviates the special development of genetic populations, which is particularly difficult for long-lived tree crops. PMID:27085183

  20. Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin

    Science.gov (United States)

    Zhu, Lixin; Hatakeyama, Jason; Zhang, Bing; Makdisi, Joy; Ender, Cody; Forte, John G.

    2009-01-01

    ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an increased gradient of expression from the neck to the base of the glands. In addition, the staining pattern of moesin revealed a branched morphology for the gastric lumen. This pattern of short branches extending from the glandular lumen was confirmed by using antibody against zonula occludens-1 (ZO-1) to stain tight junctions. With a mucous neck cell probe (lectin GSII, from Griffonia simplicifolia) and a chief cell marker (pepsinogen C), immunohistochemistry revealed that the mucous neck cells at the top of the glands do not express moesin, but, progressing toward the base, mucous cells showing decreased GSII staining had low or moderate level of moesin expression. The level of moesin expression continued to increase toward the base of the glands and reached a plateau in the base where chief cells and parietal cells abound. The level of pepsinogen expression also increased toward the base. Pepsinogen C was located on cytoplasmic granules and/or more generally distributed in chief cells, whereas moesin was exclusively expressed on the apical membrane. This is a clear demonstration of distinctive cellular expression of two ERM family members in the same tissue. The results provide the first evidence that moesin is involved in the cell biology of chief cells. Novel insights on gastric gland morphology revealed by the moesin and ZO-1 staining provide the basis for a model of cell maturation and migration within the gland. PMID:19074636

  1. Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin.

    Science.gov (United States)

    Zhu, Lixin; Hatakeyama, Jason; Zhang, Bing; Makdisi, Joy; Ender, Cody; Forte, John G

    2009-02-01

    ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an increased gradient of expression from the neck to the base of the glands. In addition, the staining pattern of moesin revealed a branched morphology for the gastric lumen. This pattern of short branches extending from the glandular lumen was confirmed by using antibody against zonula occludens-1 (ZO-1) to stain tight junctions. With a mucous neck cell probe (lectin GSII, from Griffonia simplicifolia) and a chief cell marker (pepsinogen C), immunohistochemistry revealed that the mucous neck cells at the top of the glands do not express moesin, but, progressing toward the base, mucous cells showing decreased GSII staining had low or moderate level of moesin expression. The level of moesin expression continued to increase toward the base of the glands and reached a plateau in the base where chief cells and parietal cells abound. The level of pepsinogen expression also increased toward the base. Pepsinogen C was located on cytoplasmic granules and/or more generally distributed in chief cells, whereas moesin was exclusively expressed on the apical membrane. This is a clear demonstration of distinctive cellular expression of two ERM family members in the same tissue. The results provide the first evidence that moesin is involved in the cell biology of chief cells. Novel insights on gastric gland morphology revealed by the moesin and ZO-1 staining provide the basis for a model of cell maturation and migration within the gland. PMID:19074636

  2. Adaptation to Real Motion Reveals Direction-selective Interactions between Real and Implied Motion Processing

    NARCIS (Netherlands)

    Lorteije, Jeannette A.M.; Kenemans, Leon; Jellema, Tjeerd; Lubbe, van der Rob H.J.; Lommers, Marjolein W.; Wezel, van Richard J.A.

    2007-01-01

    Viewing static pictures of running humans evokes neural activity in the dorsal motion-sensitive cortex. To establish whether this response arises from direction-selective neurons that are also involved in real motion processing, we measured the visually evoked potential to implied motion following a

  3. Adaptation to real motion reveals direction-selective interactions between real and implied motion processing

    NARCIS (Netherlands)

    Lorteije, J.A.M.; Kenemans, J.L.; Jellema, T.; Lubbe, R.H.J. van der; Lommers, M.W.; Wezel, R.J.A. van

    2007-01-01

    Viewing static pictures of running humans evokes neural activity in the dorsal motion-sensitive cortex. To establish whether this response arises from direction-selective neurons that are also involved in real motion processing, we measured the visually evoked potential to implied motion following a

  4. Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks.

    Science.gov (United States)

    Yang, Hee-Jeong; Bogomolnaya, Lydia M; Elfenbein, Johanna R; Endicott-Yazdani, Tiana; Reynolds, M Megan; Porwollik, Steffen; Cheng, Pui; Xia, Xiao-Qin; McClelland, Michael; Andrews-Polymenis, Helene

    2016-04-01

    Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used bySalmonellato colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence ofSalmonella entericaserotype Typhimurium in chickens. A library of 182S.Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks.STM0580,STM1295,STM1297,STM3612,STM3615, andSTM3734are needed forSalmonellato colonize and persist in chicks and were not previously associated with this ability. One of these key genes,STM1297(selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

  5. A gallbladder tumor revealing metastatic clear cell renal carcinoma: report of case and review of literature

    OpenAIRE

    Ghaouti Merieme; Znati Kaoutar; Jahid Ahmed; Zouaidia Fouad; Bernoussi Zakiya; Fakir Youssef El; Mahassini Najat

    2013-01-01

    Abstract Metastatic renal cell carcinoma in the gallbladder is extremely rare, with reported frequencies of less than 0.6% in large autopsy reviews. Only 40 cases were reported in the literature. We report a first case of gallbladder polypoid tumor revealing metastatic clear cell renal cell carcinoma, which demonstrates the importance of radiological tests, histology and immunohistochemistry when making a definitive diagnosis. These examinations also allow differentiating metastatic clear cel...

  6. A Survey on Cell Selection Schemes for Femtocell Networks

    Directory of Open Access Journals (Sweden)

    Darshna Prajapati, Vineet Richhariya

    2014-07-01

    Full Text Available —With ever increasing number of mobile devices, operators have to search for new alternative to handle user demands. Deployment of additional base stations is always an option to improve coverage of capacity. However, it comes with the cost of incresed operational and capital expenditure. Interestingly, it has been seen that nearly 80% of mobile data demands are originating from indoor home and office users. To handle these indoor demands, deployment of small, low power femtocell access points have been suggested. However, in current deployment scenario, most femtocell are underutilised. To improve user association and resource utilization in femtocell, various cell selection schemes have been suggested. In this paper, we analyse various cell selction schemes available in the literature. Additionally, we look at energy efficiency aspect of these cell selection schemes.

  7. Control of Mitral/Tufted Cell Output by Selective Inhibition among Olfactory Bulb Glomeruli.

    Science.gov (United States)

    Economo, Michael N; Hansen, Kyle R; Wachowiak, Matt

    2016-07-20

    Inhibition is fundamental to information processing by neural circuits. In the olfactory bulb (OB), glomeruli are the functional units for odor information coding, but inhibition among glomeruli is poorly characterized. We used two-photon calcium imaging in anesthetized and awake mice to visualize both odorant-evoked excitation and suppression in OB output neurons (mitral and tufted, MT cells). MT cell response polarity mapped uniformly to discrete OB glomeruli, allowing us to analyze how inhibition shapes OB output relative to the glomerular map. Odorants elicited unique patterns of suppression in only a subset of glomeruli in which such suppression could be detected, and excited and suppressed glomeruli were spatially intermingled. Binary mixture experiments revealed that interglomerular inhibition could suppress excitatory mitral cell responses to odorants. These results reveal that inhibitory OB circuits nonlinearly transform odor representations and support a model of selective and nonrandom inhibition among glomerular ensembles. PMID:27346531

  8. Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity.

    Science.gov (United States)

    Chiu, Isaac M; Barrett, Lee B; Williams, Erika K; Strochlic, David E; Lee, Seungkyu; Weyer, Andy D; Lou, Shan; Bryman, Gregory S; Roberson, David P; Ghasemlou, Nader; Piccoli, Cara; Ahat, Ezgi; Wang, Victor; Cobos, Enrique J; Stucky, Cheryl L; Ma, Qiufu; Liberles, Stephen D; Woolf, Clifford J

    2014-01-01

    The somatosensory nervous system is critical for the organism's ability to respond to mechanical, thermal, and nociceptive stimuli. Somatosensory neurons are functionally and anatomically diverse but their molecular profiles are not well-defined. Here, we used transcriptional profiling to analyze the detailed molecular signatures of dorsal root ganglion (DRG) sensory neurons. We used two mouse reporter lines and surface IB4 labeling to purify three major non-overlapping classes of neurons: 1) IB4(+)SNS-Cre/TdTomato(+), 2) IB4(-)SNS-Cre/TdTomato(+), and 3) Parv-Cre/TdTomato(+) cells, encompassing the majority of nociceptive, pruriceptive, and proprioceptive neurons. These neurons displayed distinct expression patterns of ion channels, transcription factors, and GPCRs. Highly parallel qRT-PCR analysis of 334 single neurons selected by membership of the three populations demonstrated further diversity, with unbiased clustering analysis identifying six distinct subgroups. These data significantly increase our knowledge of the molecular identities of known DRG populations and uncover potentially novel subsets, revealing the complexity and diversity of those neurons underlying somatosensation. PMID:25525749

  9. Single-cell lineage tracking analysis reveals that an established cell line comprises putative cancer stem cells and their heterogeneous progeny

    Science.gov (United States)

    Sato, Sachiko; Rancourt, Ann; Sato, Yukiko; Satoh, Masahiko S.

    2016-01-01

    Mammalian cell culture has been used in many biological studies on the assumption that a cell line comprises putatively homogeneous clonal cells, thereby sharing similar phenotypic features. This fundamental assumption has not yet been fully tested; therefore, we developed a method for the chronological analysis of individual HeLa cells. The analysis was performed by live cell imaging, tracking of every single cell recorded on imaging videos, and determining the fates of individual cells. We found that cell fate varied significantly, indicating that, in contrast to the assumption, the HeLa cell line is composed of highly heterogeneous cells. Furthermore, our results reveal that only a limited number of cells are immortal and renew themselves, giving rise to the remaining cells. These cells have reduced reproductive ability, creating a functionally heterogeneous cell population. Hence, the HeLa cell line is maintained by the limited number of immortal cells, which could be putative cancer stem cells. PMID:27003384

  10. Simultaneous dual-task performance reveals parallel response selection after practice

    Science.gov (United States)

    Hazeltine, Eliot; Teague, Donald; Ivry, Richard B.

    2002-01-01

    E. H. Schumacher, T. L. Seymour, J. M. Glass, D. E. Kieras, and D. E. Meyer (2001) reported that dual-task costs are minimal when participants are practiced and give the 2 tasks equal emphasis. The present research examined whether such findings are compatible with the operation of an efficient response selection bottleneck. Participants trained until they were able to perform both tasks simultaneously without interference. Novel stimulus pairs produced no reaction time costs, arguing against the development of compound stimulus-response associations (Experiment 1). Manipulating the relative onsets (Experiments 2 and 4) and durations (Experiments 3 and 4) of response selection processes did not lead to dual-task costs. The results indicate that the 2 tasks did not share a bottleneck after practice.

  11. Ontogeny of energy allocation reveals selective pressure promoting risk-taking behaviour in young fish cohorts

    OpenAIRE

    Biro, Peter A.; Post, John R.; Abrahams, Mark V

    2005-01-01

    Given limited food, prey fishes in a temperate climate must take risks to acquire sufficient reserves for winter and/or to outgrow vulnerability to predation. However, how can we distinguish which selective pressure promotes risk-taking when larger body size is always beneficial? To address this question, we examined patterns of energy allocation in populations of age-0 trout to determine if greater risk-taking corresponds with energy allocation to lipids or to somatic growth. Trout achieved ...

  12. A trait-based approach reveals the feeding selectivity of a small endangered Mediterranean fish.

    Science.gov (United States)

    Rodríguez-Lozano, Pablo; Verkaik, Iraima; Maceda-Veiga, Alberto; Monroy, Mario; de Sostoa, Adolf; Rieradevall, Maria; Prat, Narcís

    2016-05-01

    Functional traits are growing in popularity in modern ecology, but feeding studies remain primarily rooted in a taxonomic-based perspective. However, consumers do not have any reason to select their prey using a taxonomic criterion, and prey assemblages are variable in space and time, which makes taxon-based studies assemblage-specific. To illustrate the benefits of the trait-based approach to assessing food choice, we studied the feeding ecology of the endangered freshwater fish Barbus meridionalis. We hypothesized that B. meridionalis is a selective predator which food choice depends on several prey morphological and behavioral traits, and thus, its top-down pressure may lead to changes in the functional composition of in-stream macroinvertebrate communities. Feeding selectivity was inferred by comparing taxonomic and functional composition (13 traits) between ingested and free-living potential prey using the Jacob's electivity index. Our results showed that the fish diet was influenced by 10 of the 13 traits tested. Barbus meridionalis preferred prey with a potential size of 5-10 mm, with a medium-high drift tendency, and that drift during daylight. Potential prey with no body flexibility, conical shape, concealment traits (presence of nets and/or cases, or patterned coloration), and high aggregation tendency had a low predation risk. Similarly, surface swimmers and interstitial taxa were low vulnerable to predation. Feeding selectivity altered the functional composition of the macroinvertebrate communities. Fish absence favored taxa with weak aggregation tendency, weak flexibility, and a relatively large size (10-20 mm of potential size). Besides, predatory invertebrates may increase in fish absence. In conclusion, our study shows that the incorporation of the trait-based approach in diet studies is a promising avenue to improve our mechanistic understanding of predator-prey interactions and to help predict the ecological outcomes of predator invasions and

  13. Selection on Human Genes as Revealed by Comparisons to Chimpanzee cDNA

    OpenAIRE

    Hellmann, Ines; Zöllner, Sebastian; Enard, Wolfgang; Ebersberger , Ingo; Nickel, Birgit; Pääbo, Svante

    2003-01-01

    To better understand the evolutionary forces that affect human genes, we sequenced 5055 expressed sequence tags from the chimpanzee and compared them to their human counterparts. In conjunction with intergenic chimpanzee DNA sequences and data on human single-nucleotide polymorphisms in the genes studied, this allows us to gauge the extent to which selection affects human genes at a genome-wide scale. The comparison to intergenic DNA sequences indicates that about 39% of silent sites in prote...

  14. Complex Selection on Human Polyadenylation Signals Revealed by Polymorphism and Divergence Data

    Science.gov (United States)

    Kainov, Yaroslav A.; Aushev, Vasily N.; Naumenko, Sergey A.; Tchevkina, Elena M.; Bazykin, Georgii A.

    2016-01-01

    Polyadenylation is a step of mRNA processing which is crucial for its expression and stability. The major polyadenylation signal (PAS) represents a nucleotide hexamer that adheres to the AATAAA consensus sequence. Over a half of human genes have multiple cleavage and polyadenylation sites, resulting in a great diversity of transcripts differing in function, stability, and translational activity. Here, we use available whole-genome human polymorphism data together with data on interspecies divergence to study the patterns of selection acting on PAS hexamers. Common variants of PAS hexamers are depleted of single nucleotide polymorphisms (SNPs), and SNPs within PAS hexamers have a reduced derived allele frequency (DAF) and increased conservation, indicating prevalent negative selection; at the same time, the SNPs that “improve” the PAS (i.e., those leading to higher cleavage efficiency) have increased DAF, compared to those that “impair” it. SNPs are rarer at PAS of “unique” polyadenylation sites (one site per gene); among alternative polyadenylation sites, at the distal PAS and at exonic PAS. Similar trends were observed in DAFs and divergence between species of placental mammals. Thus, selection permits PAS mutations mainly at redundant and/or weakly functional PAS. Nevertheless, a fraction of the SNPs at PAS hexamers likely affect gene functions; in particular, some of the observed SNPs are associated with disease. PMID:27324920

  15. Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq.

    Science.gov (United States)

    Gokce, Ozgun; Stanley, Geoffrey M; Treutlein, Barbara; Neff, Norma F; Camp, J Gray; Malenka, Robert C; Rothwell, Patrick E; Fuccillo, Marc V; Südhof, Thomas C; Quake, Stephen R

    2016-07-26

    The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs) that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states. PMID:27425622

  16. Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq

    Directory of Open Access Journals (Sweden)

    Ozgun Gokce

    2016-07-01

    Full Text Available The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states.

  17. Sodium selectivity of semicircular canal duct epithelial cells

    Directory of Open Access Journals (Sweden)

    Harbidge Donald G

    2011-09-01

    Full Text Available Abstract Background Sodium absorption by semicircular canal duct (SCCD epithelial cells is thought to contribute to the homeostasis of the volume of vestibular endolymph. It was previously shown that the epithelial cells could absorb Na+ under control of a glucocorticoid hormone (dexamethasone and the absorptive transepithelial current was blocked by amiloride. The most commonly-observed target of amiloride is the epithelial sodium channel (ENaC, comprised of the three subunits α-, β- and γ-ENaC. However, other cation channels have also been observed to be sensitive in a similar concentration range. The aim of this study was to determine whether SCCD epithelial cells absorb only Na+ or also K+ through an amiloride-sensitive pathway. Parasensory K+ absorption could contribute to regulation of the transduction current through hair cells, as found to occur via vestibular transitional cells [S. H. Kim and D. C. Marcus. Regulation of sodium transport in the inner ear. Hear.Res. doi:10.1016/j.heares.2011.05.003, 2011]. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6197, whole-cell patch clamp and transepithelial recordings in primary cultures of rat SCCD. α-, β- and γ-ENaC were all previously reported as present. The selectivity of the amiloride-sensitive transepithelial and cell membrane currents was observed in Ussing chamber and whole-cell patch clamp recordings. The cell membrane currents were carried by Na+ but not K+, but the Na+ selectivity disappeared when the cells were cultured on impermeable supports. Transepithelial currents across SCCD were also carried exclusively by Na+. Conclusions These results are consistent with the amiloride-sensitive absorptive flux of SCCD mediated by a highly Na+-selective channel, likely αβγ-ENaC. These epithelial cells therefore absorb only Na+ via the amiloride-sensitive pathway and do not provide a parasensory K+ efflux from the

  18. Patterns of cell division revealed by transcriptional regulation of genes during the cell cycle in plants.

    OpenAIRE

    Fobert, P R; Coen, E S; Murphy, G. J.; Doonan, J H

    1994-01-01

    Transcripts from five cell cycle related genes accumulate in isolated cells dispersed throughout the actively dividing regions of plant meristems. We propose that this pattern reflects gene expression during particular phases of the cell division cycle. The high proportion of isolated cells suggests that synchrony between daughter cells is rapidly lost following mitosis. This is the first time that such a cell specific expression pattern has been described in a higher organism. Counterstainin...

  19. Live cell linear dichroism imaging reveals extensive membrane ruffling within the docking structure of natural killer cell immune synapses

    DEFF Research Database (Denmark)

    Benninger, Richard K P; Vanherberghen, Bruno; Young, Stephen;

    2009-01-01

    We have applied fluorescence imaging of two-photon linear dichroism to measure the subresolution organization of the cell membrane during formation of the activating (cytolytic) natural killer (NK) cell immune synapse (IS). This approach revealed that the NK cell plasma membrane is convoluted into...... absent from the center of the mature synapse. Understanding the role of such extensive membrane ruffling in the assembly of cytolytic synapses is an intriguing new goal....

  20. Cell surface thiol isomerases may explain the platelet-selective action of S-nitrosoglutathione.

    Science.gov (United States)

    Xiao, Fang; Gordge, Michael P

    2011-10-30

    S-nitrosoglutathione (GSNO) at low concentration inhibits platelet aggregation without causing vasodilation, suggesting platelet-selective nitric oxide delivery. The mechanism of this selectivity is unknown, but may involve cell surface thiol isomerases, in particular protein disulphide isomerase (csPDI) (EC 5.3.4.1). We have now compared csPDI expression and activity on platelets, endothelial cells and vascular smooth muscle cells, and the dependence on thiol reductase activity of these cell types for NO uptake from GSNO. csPDI expression was measured by flow cytometry and its reductase activity using the pseudosubstrate dieosin glutathione disulphide. This activity assay was adapted and validated for 96-well plate format. Flow cytometry revealed csPDI on all three cell types, but percentage positivity of expression was higher on platelets than on vascular cells. Consistent with this, thiol isomerase-related reductase activity was higher on platelets (Pionomycin) increased csPDI activity on both platelets and smooth muscle cells, but not on endothelium. Intracellular NO delivery from GSNO was greater in platelets than in vascular cells (Pselective actions of GSNO and help define its antithrombotic potential. PMID:21642008

  1. Breeding signature of combining ability improvement revealed by a genomic variation map from recurrent selection population in Brassica napus

    Science.gov (United States)

    Zhao, Xinwang; Li, Bao; Zhang, Ka; Hu, Kaining; Yi, Bin; Wen, Jing; Ma, Chaozhi; Shen, Jinxiong; Fu, Tingdong; Tu, Jinxing

    2016-01-01

    Combining ability is crucial for parent selection in crop hybrid breeding. The present investigation and results had revealed the underlying genetic factors which might contribute in adequate combining ability, further assisting in enhancing heterosis and stability. Here, we conducted a large-scale analysis of genomic variation in order to define genomic regions affecting the combining ability in recurrent selection population of rapeseed. A population of 175 individuals was genotyped with the Brassica60K SNP chip. 525 hybrids were assembled with three different testers and used to evaluate the general combining ability (GCA) in three environments. By detecting the changes of the genomic variation, we identified 376 potential genome regions, spanning 3.03% of rapeseed genome which provided QTL-level resolution on potentially selected variants. More than 96% of these regions were located in the C subgenome, indicating that C subgenome had sustained stronger selection pressure in the breeding program than the A subgenome. In addition, a high level of linkage disequilibrium in rapeseed genome was detected, suggesting that marker-assisted selection for the population improvement might be easily implemented. This study outlines the evidence for high GCA on a genomic level and provided underlying molecular mechanism for recurrent selection improvement in B. napus. PMID:27412721

  2. Breeding signature of combining ability improvement revealed by a genomic variation map from recurrent selection population in Brassica napus.

    Science.gov (United States)

    Zhao, Xinwang; Li, Bao; Zhang, Ka; Hu, Kaining; Yi, Bin; Wen, Jing; Ma, Chaozhi; Shen, Jinxiong; Fu, Tingdong; Tu, Jinxing

    2016-01-01

    Combining ability is crucial for parent selection in crop hybrid breeding. The present investigation and results had revealed the underlying genetic factors which might contribute in adequate combining ability, further assisting in enhancing heterosis and stability. Here, we conducted a large-scale analysis of genomic variation in order to define genomic regions affecting the combining ability in recurrent selection population of rapeseed. A population of 175 individuals was genotyped with the Brassica60K SNP chip. 525 hybrids were assembled with three different testers and used to evaluate the general combining ability (GCA) in three environments. By detecting the changes of the genomic variation, we identified 376 potential genome regions, spanning 3.03% of rapeseed genome which provided QTL-level resolution on potentially selected variants. More than 96% of these regions were located in the C subgenome, indicating that C subgenome had sustained stronger selection pressure in the breeding program than the A subgenome. In addition, a high level of linkage disequilibrium in rapeseed genome was detected, suggesting that marker-assisted selection for the population improvement might be easily implemented. This study outlines the evidence for high GCA on a genomic level and provided underlying molecular mechanism for recurrent selection improvement in B. napus. PMID:27412721

  3. Selective laser removal of the dimer layer from Si(100) surfaces revealed by scanning tunneling microscopy

    International Nuclear Information System (INIS)

    Scanning tunneling microscopy (STM) of laser-irradiated Si(100) surfaces shows that the dimerized outermost layer can be selectively removed by a pulsed Nd:YAG laser with a fluence below the melt threshold. The atoms in the laser-uncovered second layer are close to positions of a bulk terminated (1x1) structure, but with a slight pairing, while dimers retain a (2x1) configuration in the first layer. The pairing distance and fraction of the remaining dimers decrease with increasing laser exposures. The laser-uncovered layer also remains free of vacancies. copyright 1996 The American Physical Society

  4. Monitoring of Water Spectral Pattern Reveals Differences in Probiotics Growth When Used for Rapid Bacteria Selection.

    Directory of Open Access Journals (Sweden)

    Aleksandar Slavchev

    Full Text Available Development of efficient screening method coupled with cell functionality evaluation is highly needed in contemporary microbiology. The presented novel concept and fast non-destructive method brings in to play the water spectral pattern of the solution as a molecular fingerprint of the cell culture system. To elucidate the concept, NIR spectroscopy with Aquaphotomics were applied to monitor the growth of sixteen Lactobacillus bulgaricus one Lactobacillus pentosus and one Lactobacillus gasseri bacteria strains. Their growth rate, maximal optical density, low pH and bile tolerances were measured and further used as a reference data for analysis of the simultaneously acquired spectral data. The acquired spectral data in the region of 1100-1850nm was subjected to various multivariate data analyses - PCA, OPLS-DA, PLSR. The results showed high accuracy of bacteria strains classification according to their probiotic strength. Most informative spectral fingerprints covered the first overtone of water, emphasizing the relation of water molecular system to cell functionality.

  5. Selective Induction of Cancer Cell Death by Targeted Granzyme B

    Directory of Open Access Journals (Sweden)

    Robert A. Jabulowsky

    2013-02-01

    Full Text Available The potential utility of immunotoxins for cancer therapy has convincingly been demonstrated in clinical studies. Nevertheless, the high immunogenicity of their bacterial toxin domain represents a critical limitation, and has prompted the evaluation of cell-death inducing proteins of human origin as a basis for less immunogenic immunotoxin-like molecules. In this review, we focus on the current status and future prospects of targeted fusion proteins for cancer therapy that employ granzyme B (GrB from cytotoxic lymphocytes as a cytotoxic moiety. Naturally, this serine protease plays a critical role in the immune defense by inducing apoptotic target cell death upon cleavage of intracellular substrates. Advances in understanding of the structure and function of GrB enabled the generation of chimeric fusion proteins that carry a heterologous cell binding domain for recognition of tumor-associated cell surface antigens. These hybrid molecules display high selectivity for cancer cells, with cell killing activities similar to that of corresponding recombinant toxins. Recent findings have helped to understand and circumvent intrinsic cell binding of GrB and susceptibility of the enzyme to inhibition by serpins. This now allows the rational design of optimized GrB derivatives that avoid sequestration by binding to non-target tissues, limit off-target effects, and overcome resistance mechanisms in tumor cells.

  6. Conformational Selection in a Protein-Protein Interaction Revealed by Dynamic Pathway Analysis

    Directory of Open Access Journals (Sweden)

    Kalyan S. Chakrabarti

    2016-01-01

    Full Text Available Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection or are caused in response to ligand binding (induced fit. Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here, we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using nuclear magnetic resonance (NMR spectroscopy, stopped-flow kinetics, and isothermal titration calorimetry, we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Protein dynamics in free recoverin limits the overall rate of binding.

  7. Conformational Selection in a Protein-Protein Interaction Revealed by Dynamic Pathway Analysis.

    Science.gov (United States)

    Chakrabarti, Kalyan S; Agafonov, Roman V; Pontiggia, Francesco; Otten, Renee; Higgins, Matthew K; Schertler, Gebhard F X; Oprian, Daniel D; Kern, Dorothee

    2016-01-01

    Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here, we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using nuclear magnetic resonance (NMR) spectroscopy, stopped-flow kinetics, and isothermal titration calorimetry, we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Protein dynamics in free recoverin limits the overall rate of binding. PMID:26725117

  8. Selective Antitumor Activity of Ibrutinib in EGFR-Mutant Non–Small Cell Lung Cancer Cells

    OpenAIRE

    Gao, Wen; Wang, Michael; Wang, Li; Lu, Haibo; Wu, Shuhong; Dai, Bingbing; Ou, Zhishuo; Zhang, Liang; Heymach, John V.; Gold, Kathryn A.; Minna, John ,; Roth, Jack A.; Hofstetter, Wayne L.; Swisher, Stephen G.; Fang, Bingliang

    2014-01-01

    Ibrutinib, which irreversibly inhibits Bruton tyrosine kinase, was evaluated for antitumor activity in a panel of non–small cell lung cancer (NSCLC) cell lines and found to selectively inhibit growth of NSCLC cells carrying mutations in the epidermal growth factor receptor (EGFR) gene, including T790M mutant and erlotinib-resistant H1975 cells. Ibrutinib induced dose-dependent inhibition of phosphor-EGFR at both Y1068 and Y1173 sites, suggesting ibrutinib functions as an EGFR inhibitor. Survi...

  9. A transgenic mouse marking live replicating cells reveals in vivo transcriptional program of proliferation

    DEFF Research Database (Denmark)

    Klochendler, Agnes; Weinberg-Corem, Noa; Moran, Maya;

    2012-01-01

    biological material. We describe a transgenic mouse strain, expressing a CyclinB1-GFP fusion reporter, that marks replicating cells in the S/G2/M phases of the cell cycle. Using flow cytometry, we isolate live replicating cells from the liver and compare their transcriptome to that of quiescent cells to......Most adult mammalian tissues are quiescent, with rare cell divisions serving to maintain homeostasis. At present, the isolation and study of replicating cells from their in vivo niche typically involves immunostaining for intracellular markers of proliferation, causing the loss of sensitive...... reveal gene expression programs associated with cell proliferation in vivo. We find that replicating hepatocytes have reduced expression of genes characteristic of liver differentiation. This reporter system provides a powerful platform for gene expression and metabolic and functional studies of...

  10. Metabolomics reveals mycoplasma contamination interferes with the metabolism of PANC-1 cells.

    Science.gov (United States)

    Yu, Tao; Wang, Yongtao; Zhang, Huizhen; Johnson, Caroline H; Jiang, Yiming; Li, Xiangjun; Wu, Zeming; Liu, Tian; Krausz, Kristopher W; Yu, Aiming; Gonzalez, Frank J; Huang, Min; Bi, Huichang

    2016-06-01

    Mycoplasma contamination is a common problem in cell culture and can alter cellular functions. Since cell metabolism is either directly or indirectly involved in every aspect of cell function, it is important to detect changes to the cellular metabolome after mycoplasma infection. In this study, liquid chromatography mass spectrometry (LC/MS)-based metabolomics was used to investigate the effect of mycoplasma contamination on the cellular metabolism of human pancreatic carcinoma cells (PANC-1). Multivariate analysis demonstrated that mycoplasma contamination induced significant metabolic changes in PANC-1 cells. Twenty-three metabolites were identified and found to be involved in arginine and purine metabolism and energy supply. This study demonstrates that mycoplasma contamination significantly alters cellular metabolite levels, confirming the compelling need for routine checking of cell cultures for mycoplasma contamination, particularly when used for metabolomics studies. Graphical abstract Metabolomics reveals mycoplasma contamination changes the metabolome of PANC-1 cells. PMID:27074779

  11. Single-Cell Transcriptome Analyses Reveal Signals to Activate Dormant Neural Stem Cells

    OpenAIRE

    Luo, Yuping; Coskun, Volkan; Liang, Aibing; Yu, Juehua; Cheng, Liming; Ge, Weihong; Shi, Zhanping; Zhang, Kunshan; Li, Chun; Cui, Yaru; Lin, Haijun; Luo, Dandan; Wang, Junbang; Lin, Connie; Dai, Zachary

    2015-01-01

    The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133+/GFAP− ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133+/GFAP− quiescent cells were enriched...

  12. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    Science.gov (United States)

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  13. Building Cell Selectivity into CPP-Mediated Strategies

    Directory of Open Access Journals (Sweden)

    Irene Martín

    2010-05-01

    Full Text Available There is a pressing need for more effective and selective therapies for cancer and other diseases. Consequently, much effort is being devoted to the development of alternative experimental approaches based on selective systems, which are designed to be specifically directed against target cells. In addition, a large number of highly potent therapeutic molecules are being discovered. However, they do not reach clinical trials because of their low delivery, poor specificity or their incapacity to bypass the plasma membrane. Cell-penetrating peptides (CPPs are an open door for cell-impermeable compounds to reach intracellular targets. Putting all these together, research is sailing in the direction of the design of systems with the capacity to transport new drugs into a target cell. Some CPPs show cell type specificity while others require modifications or form part of more sophisticated drug delivery systems. In this review article we summarize several strategies for directed drug delivery involving CPPs that have been reported in the literature.

  14. Triclosan Resistome from Metagenome Reveals Diverse Enoyl Acyl Carrier Protein Reductases and Selective Enrichment of Triclosan Resistance Genes.

    Science.gov (United States)

    Khan, Raees; Kong, Hyun Gi; Jung, Yong-Hoon; Choi, Jinhee; Baek, Kwang-Yeol; Hwang, Eul Chul; Lee, Seon-Woo

    2016-01-01

    Triclosan (TCS) is a widely used antimicrobial agent and TCS resistance is considered to have evolved in diverse organisms with extensive use of TCS, but distribution of TCS resistance has not been well characterized. Functional screening of the soil metagenome in this study has revealed that a variety of target enoyl acyl carrier protein reductases (ENR) homologues are responsible for the majority of TCS resistance. Diverse ENRs similar to 7-α-hydroxysteroid dehydrogenase (7-α-HSDH), FabG, or the unusual YX7K-type ENR conferred extreme tolerance to TCS. The TCS-refractory 7-α HSDH-like ENR and the TCS-resistant YX7K-type ENR seem to be prevalent in human pathogenic bacteria, suggesting that a selective enrichment occurred in pathogenic bacteria in soil. Additionally, resistance to multiple antibiotics was found to be mediated by antibiotic resistance genes that co-localize with TCS resistance determinants. Further comparative analysis of ENRs from 13 different environments has revealed a huge diversity of both prototypic and metagenomic TCS-resistant ENRs, in addition to a selective enrichment of TCS-resistant specific ENRs in presumably TCS-contaminated environments with reduced ENR diversity. Our results suggest that long-term extensive use of TCS can lead to the selective emergence of TCS-resistant bacterial pathogens, possibly with additional resistance to multiple antibiotics, in natural environments. PMID:27577999

  15. Triclosan Resistome from Metagenome Reveals Diverse Enoyl Acyl Carrier Protein Reductases and Selective Enrichment of Triclosan Resistance Genes

    Science.gov (United States)

    Khan, Raees; Kong, Hyun Gi; Jung, Yong-Hoon; Choi, Jinhee; Baek, Kwang-Yeol; Hwang, Eul Chul; Lee, Seon-Woo

    2016-01-01

    Triclosan (TCS) is a widely used antimicrobial agent and TCS resistance is considered to have evolved in diverse organisms with extensive use of TCS, but distribution of TCS resistance has not been well characterized. Functional screening of the soil metagenome in this study has revealed that a variety of target enoyl acyl carrier protein reductases (ENR) homologues are responsible for the majority of TCS resistance. Diverse ENRs similar to 7-α-hydroxysteroid dehydrogenase (7-α-HSDH), FabG, or the unusual YX7K-type ENR conferred extreme tolerance to TCS. The TCS-refractory 7-α HSDH-like ENR and the TCS-resistant YX7K-type ENR seem to be prevalent in human pathogenic bacteria, suggesting that a selective enrichment occurred in pathogenic bacteria in soil. Additionally, resistance to multiple antibiotics was found to be mediated by antibiotic resistance genes that co-localize with TCS resistance determinants. Further comparative analysis of ENRs from 13 different environments has revealed a huge diversity of both prototypic and metagenomic TCS-resistant ENRs, in addition to a selective enrichment of TCS-resistant specific ENRs in presumably TCS-contaminated environments with reduced ENR diversity. Our results suggest that long-term extensive use of TCS can lead to the selective emergence of TCS-resistant bacterial pathogens, possibly with additional resistance to multiple antibiotics, in natural environments. PMID:27577999

  16. Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding.

    Science.gov (United States)

    Rocheleau, Gail; Petrillo, Jessica; Guogas, Laura; Gehrke, Lee

    2004-08-01

    The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity. PMID:15254175

  17. Selective-emitter crystalline silicon solar cells using phosphorus paste

    International Nuclear Information System (INIS)

    Selective-emitter structures have been studied to improve the conversion efficiency of crystalline silicon solar cells. However, such structures require additional complicated processes and incur extra cost. In this work, we used phosphorus paste (P-paste) to form a heavily-doped region beneath the grid and POCl3 to create a shallow emitter area. This method should be convenient to use in the solar-cell industry because it requires only additional P paste printing, compared to the case of homogeneous solar cells. Diffusion parameters including the temperature, diffusion time, and ambient gases were optimized. We observed that the spreading of the P paste was affected by the pyramidal size of the textured wafer due to the low viscosity of the P paste. The pyramidal height of the textured silicon surface was optimized at 3 μm to counterbalance the surface reflectance and the spreading of the P paste. The short-circuit current density of the completed selective emitter solar cell was increased, and an improvement of blue response in the internal quantum efficiency was seen while contact properties such as the fill factor deteriorated due to the spreading of the P paste and the thin emitter on top of the pyramid of the textured silicon surface. Double printing of the P paste was applied to solve this contact problem; a fill factor improvement of 2.4% was obtained.

  18. Selective-emitter crystalline silicon solar cells using phosphorus paste

    Science.gov (United States)

    Jeong, Kyung Taek; Kang, Min Gu; Song, Hee-eun

    2014-11-01

    Selective-emitter structures have been studied to improve the conversion efficiency of crystalline silicon solar cells. However, such structures require additional complicated processes and incur extra cost. In this work, we used phosphorus paste (P-paste) to form a heavily-doped region beneath the grid and POCl3 to create a shallow emitter area. This method should be convenient to use in the solar-cell industry because it requires only additional P paste printing, compared to the case of homogeneous solar cells. Diffusion parameters including the temperature, diffusion time, and ambient gases were optimized. We observed that the spreading of the P paste was affected by the pyramidal size of the textured wafer due to the low viscosity of the P paste. The pyramidal height of the textured silicon surface was optimized at 3 μm to counterbalance the surface reflectance and the spreading of the P paste. The short-circuit current density of the completed selective emitter solar cell was increased, and an improvement of blue response in the internal quantum efficiency was seen while contact properties such as the fill factor deteriorated due to the spreading of the P paste and the thin emitter on top of the pyramid of the textured silicon surface. Double printing of the P paste was applied to solve this contact problem; a fill factor improvement of 2.4% was obtained.

  19. Selective Interlayers and Contacts in Organic Photovoltaic Cells.

    Science.gov (United States)

    Ratcliff, Erin L; Zacher, Brian; Armstrong, Neal R

    2011-06-01

    Organic photovoltaic cells (OPVs) are promising solar electric energy conversion systems with impressive recent optimization of active layers. OPV optimization must now be accompanied by the development of new charge-selective contacts and interlayers. This Perspective considers the role of interface science in energy harvesting using OPVs, looking back at early photoelectrochemical (photogalvanic) energy conversion platforms, which suffered from a lack of charge carrier selectivity. We then examine recent platforms and the fundamental aspects of selective harvesting of holes and electrons at opposite contacts. For blended heterojunction OPVs, contact/interlayer design is especially critical because charge harvesting competes with recombination at these same contacts. New interlayer materials can modify contacts to both control work function and introduce selectivity and chemical compatibility with nonpolar active layers and add thermodynamic and kinetic selectivity to charge harvesting. We briefly discuss the surface and interface science required for the development of new interlayer materials and take a look ahead at the challenges yet to be faced in their optimization. PMID:26295432

  20. Kinetic modeling reveals a common death niche for newly formed and mature B cells.

    Directory of Open Access Journals (Sweden)

    Gitit Shahaf

    Full Text Available BACKGROUND: B lymphocytes are subject to elimination following strong BCR ligation in the absence of appropriate second signals, and this mechanism mediates substantial cell losses during late differentiation steps in the bone marrow and periphery. Mature B cells may also be eliminated through this mechanism as well as through normal turnover, but the population containing mature cells destined for elimination has not been identified. Herein, we asked whether the transitional 3 (T3 subset, which contains most newly formed cells undergoing anergic death, could also include mature B cells destined for elimination. METHODOLOGY/PRINCIPAL FINDINGS: To interrogate this hypothesis and its implications, we applied mathematical models to previously generated in vivo labeling data. Our analyses reveal that the death rate of T3 B cells is far higher than the death rates of all other splenic B cell subpopulations. Further, the model, in which the T3 pool includes both newly formed and mature primary B cells destined for apoptotic death, shows that this cell loss may account for nearly all mature B cell turnover. CONCLUSIONS/SIGNIFICANCE: This finding has implications for the mechanism of normal mature B cell turnover.

  1. Collateral methotrexate resistance in cisplatin-selected murine leukemia cells

    Directory of Open Access Journals (Sweden)

    Bhushan A.

    1999-01-01

    Full Text Available Resistance to anticancer drugs is a major cause of failure of many therapeutic protocols. A variety of mechanisms have been proposed to explain this phenomenon. The exact mechanism depends upon the drug of interest as well as the tumor type treated. While studying a cell line selected for its resistance to cisplatin we noted that the cells expressed a >25,000-fold collateral resistance to methotrexate. Given the magnitude of this resistance we elected to investigate this intriguing collateral resistance. From a series of investigations we have identified an alteration in a membrane protein of the resistant cell as compared to the sensitive cells that could be the primary mechanism of resistance. Our studies reviewed here indicate decreased tyrosine phosphorylation of a protein (molecular mass = 66 in the resistant cells, which results in little or no transfer of methotrexate from the medium into the cell. Since this is a relatively novel function for tyrosine phosphorylation, this information may provide insight into possible pharmacological approaches to modify therapeutic regimens by analyzing the status of this protein in tumor samples for a better survival of the cancer patients.

  2. Characterization of Rat Hair Follicle Stem Cells Selected by Vario Magnetic Activated Cell Sorting System

    International Nuclear Information System (INIS)

    Hair follicle stem cells (HfSCs) play crucial roles in hair follicle morphogenesis and hair cycling. These stem cells are self-renewable and have the multi-lineage potential to generate epidermis, sebaceous glands, and hair follicle. The separation and identification of hair follicle stem cells are important for further research in stem cell biology. In this study, we report on the successful enrichment of rat hair follicle stem cells through vario magnetic activated cell sorting (Vario MACS) and the biological characteristics of the stem cells. We chose the HfSCs positive surface markers CD34, α6-integrin and the negative marker CD71 to design four isolation strategies: positive selection with single marker of CD34, positive selection with single marker of α6-integrin, CD71 depletion followed by CD34 positive selection, and CD71 depletion followed by α6-integrin positive selection. The results of flow cytometry analysis showed that all four strategies had ideal effects. Specifically, we conducted a series of researches on HfSCs characterized by their high level of CD34, termed CD34bri cells, and low to undetectable expression of CD34, termed CD34dim cells. CD34bri cells had greater proliferative potential and higher colony-forming ability than CD34dim cells. Furthermore, CD34bri cells had some typical characteristics as progenitor cells, such as large nucleus, obvious nucleolus, large nuclear:cytoplasmic ratio and few cytoplasmic organelles. Our findings clearly demonstrated that HfSCs with high purity and viability could be successfully enriched with Vario MACS

  3. Selective cell targeting and lineage tracing of human induced pluripotent stem cells using recombinant avian retroviruses.

    Science.gov (United States)

    Hildebrand, Laura; Seemann, Petra; Kurtz, Andreas; Hecht, Jochen; Contzen, Jörg; Gossen, Manfred; Stachelscheid, Harald

    2015-12-01

    Human induced pluripotent stem cells (hiPSC) differentiate into multiple cell types. Selective cell targeting is often needed for analyzing gene function by overexpressing proteins in a distinct population of hiPSC-derived cell types and for monitoring cell fate in response to stimuli. However, to date, this has not been possible, as commonly used viruses enter the hiPSC via ubiquitously expressed receptors. Here, we report for the first time the application of a heterologous avian receptor, the tumor virus receptor A (TVA), to selectively transduce TVA(+) cells in a mixed cell population. Expression of the TVA surface receptor via genetic engineering renders cells susceptible for infection by avian leucosis virus (ALV). We generated hiPSC lines with this stably integrated, ectopic TVA receptor gene that expressed the receptor while retaining pluripotency. The undifferentiated hiPSC(TVA+) as well as their differentiating progeny could be infected by recombinant ALV (so-called RCAS virus) with high efficiency. Due to incomplete receptor blocking, even sequential infection of differentiating or undifferentiated TVA(+) cells was possible. In conclusion, the TVA/RCAS system provides an efficient and gentle gene transfer system for hiPSC and extends our possibilities for selective cell targeting and lineage tracing studies. PMID:26109426

  4. Global discovery of erythroid long noncoding RNAs reveals novel regulators of red cell maturation

    OpenAIRE

    Alvarez-Dominguez, Juan R.; Hu, Wenqian; Yuan, Bingbing; Shi, Jiahai; Park, Staphany S.; Gromatzky, Austin A.; van Oudenaarden, Alexander; Lodish, Harvey F.

    2014-01-01

    Global lncRNA discovery reveals novel erythroid-specific lncRNAs that are dynamically expressed and targeted by GATA1, TAL1, and KLF1.Multiple types of lncRNAs promote red cell maturation by regulating neighboring loci, including DLEU2 and a novel Band 3 enhancer lncRNA.

  5. Structures of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control

    OpenAIRE

    Burke, Jason R.; Hura, Greg L.; Rubin, Seth M.

    2012-01-01

    Rubin and colleagues describe the first structures of full-length and phosphorylated Retinoblastoma (Rb) protein. These structures reveal the mechanism of Rb inactivation and provide valuable insight into this critical tumor suppressor protein's allosteric inhibition via multisite Cdk phosphorylation and its E2F and cell cycle regulation.

  6. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, Michael G.; Donohoe, Byron; Ciesielski, Peter; Nill, Jennifer; McKinney, Kellene; Mittal, Ashutosh; Katahira, Rui; Himmel, Michael; Biddy, Mary; Beckham, Gregg; Decker, Steve

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution.

  7. Surface-enhanced Raman scattering reveals adsorption of mitoxantrone on plasma membrane of living cells

    International Nuclear Information System (INIS)

    Surface-enhanced Raman scattering (SERS) spectroscopy was applied to analyze mitoxantrone (MTX) adsorption on the plasma membrane microenvironment of sensitive (HCT-116 S) or BCRP/MXR-type resistant (HCT-116 R) cells. The addition of silver colloid to MTX-treated cells revealed an enhanced Raman scattering of MTX. Addition of extracellular DNA induced a total extinction of MTX Raman intensity for both cell lines, which revealed an adsorption of MTX on plasma membrane. A threefold higher MTX Raman intensity was observed for HCT-116 R, suggesting a tight MTX adsorption in the plasma membrane microenvironment. Fluorescence confocal microscopy confirmed a relative MTX emission around plasma membrane for HCT-116 R. After 30 min at 4 deg. C, a threefold decrease of the MTX Raman scattering was observed for HCT-116 R, contrary to HCT-116 S. Permeation with benzyl alcohol revealed a threefold decrease of membrane MTX adsorption on HCT-116 R, exclusively. This additional MTX adsorption should correspond to the drug bound to an unstable site on the HCT-116 R membrane. This study showed that SERS spectroscopy could be a direct method to reveal drug adsorption to the membrane environment of living cells

  8. Principles of bacterial cell-size determination revealed by cell wall synthesis perturbations

    OpenAIRE

    Carolina Tropini; Timothy K. Lee; Jen Hsin; Samantha M. Desmarais; Tristan Ursell; Russell D. Monds; Kerwyn Casey Huang

    2014-01-01

    Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cyto...

  9. Ethanol Extracts of Selected Cyathea Species Decreased Cell Viability and Inhibited Growth in MCF 7 Cell Line Cultures.

    Science.gov (United States)

    Janakiraman, Narayanan; Johnson, Marimuthu

    2016-06-01

    Cancer is the cause of more than 6 million deaths worldwide every year. For centuries, medicinal plants have been used in the treatment of cancer. Chemotherapy, radiotherapy, surgery and acupuncture point stimulation are also used to treat cancer. The present study was intended to reveal the cytotoxic and anticancer potential of selected Cyathea species and to highlight their importance in the pharmaceutical industry for the development of cost-effective drugs. Cytotoxic studies using brine shrimp lethality bioassays and MCF 7 cell line cultures were carried out. Compared to petroleum ether, chloroform and acetone extracts, the ethanol extracts of selected Cyathea species were found to be more effective against brine shrimps. The ethanol extracts were further subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assays. A decrease in cell viability and an increase in growth inhibition were observed for the MCF 7 cell line. The maximum percentage of cell inhibition was observed in Cyathea crinit, followed by Cyathea nilgirensis and Cyathea gigantea. The results of the present study suggest that Cyathea species are an effective source of cytotoxic compounds. PMID:27342889

  10. Heterogeneity of Mesp1+ mesoderm revealed by single-cell RNA-seq.

    Science.gov (United States)

    Chan, Sunny Sun-Kin; Chan, Howe H W; Kyba, Michael

    2016-06-01

    Mesp1 is a transcription factor that promotes differentiation of pluripotent cells into different mesoderm lineages including hematopoietic, cardiac and skeletal myogenic. This occurs via at least two transient cell populations: a common hematopoietic/cardiac progenitor population and a common cardiac/skeletal myogenic progenitor population. It is not established whether Mesp1-induced mesoderm cells are intrinsically heterogeneous, or are simply capable of multiple lineage decisions. In the current study, we applied single-cell RNA-seq to analyze Mesp1+ mesoderm. Initial whole transcriptome analysis showed a surprising homogeneity among Mesp1-induced mesoderm cells. However, this apparent global homogeneity masked an intrinsic heterogeneity revealed by interrogating a panel of early mesoderm patterning factors. This approach enabled discovery of subpopulations primed for hematopoietic or cardiac development. These studies demonstrate the heterogeneic nature of Mesp1+ mesoderm. PMID:27131741

  11. Materials selection for solid oxide fuel cells (SOFCs) systems

    International Nuclear Information System (INIS)

    Because fuel cells, as electrochemical devices, convert fuels such as hydrogen into electricity without combustion they create virtually no pollution and hold the key to future prosperity and a healthy global environment. In recent years, the development and commercialisation of fuel cells systems for different applications is increasing tremendously. A discussion in made, in this paper, upon the benefits obtained from the few potential applications and the future of such devices with a particular, attention being given to design (and operation of Solid Oxide Fuel Cells (SOFCs), nothing the restrictions based on materials requirements and fuel specifications. The materials selection for this kind of fuel cells is very difficult due to the temperature range they operate in. The advantage and limitations, development state, and technical issues for novel materials used in SOFC electrodes, electrolyte and interconnect, that are the subject of a majority of contemporary research, and their properties are reviewed and discussed. A complete scenario is proposed as a competitive energy policy and a step forward to the target of sustainable development. It is emphasised that fuel cells will be a significant contributor within a portfolio of energy sources in the coming 10 to 20 years.(Author)

  12. SHAPE SELECTIVE NANOCATALYSTS FOR DIRECT METHANOL FUEL CELL APPLICATIONS

    Energy Technology Data Exchange (ETDEWEB)

    Murph, S.

    2012-09-12

    While gold and platinum have long been recognized for their beauty and value, researchers at the Savannah River National Laboratory (SRNL) are working on the nano-level to use these elements for creative solutions to our nation's energy and security needs. Multiinterdisciplinary teams consisting of chemists, materials scientists, physicists, computational scientists, and engineers are exploring unchartered territories with shape-selective nanocatalysts for the development of novel, cost effective and environmentally friendly energy solutions to meet global energy needs. This nanotechnology is vital, particularly as it relates to fuel cells.SRNL researchers have taken process, chemical, and materials discoveries and translated them for technological solution and deployment. The group has developed state-of-the art shape-selective core-shell-alloy-type gold-platinum nanostructures with outstanding catalytic capabilities that address many of the shortcomings of the Direct Methanol Fuel Cell (DMFC). The newly developed nanostructures not only busted the performance of the platinum catalyst, but also reduced the material cost and overall weight of the fuel cell.

  13. Genomic instability of micronucleated cells revealed by single-cell comparative genomic hybridization.

    NARCIS (Netherlands)

    Imle, A.; Polzer, B.; Alexander, S.; Klein, C.A.; Friedl, P.H.A.

    2009-01-01

    Nuclear variation in size and shape and genomic instability are hallmarks of dedifferentiated cancer cells. Although micronuclei are a typical long-term consequence of DNA damage, their contribution to chromosomal instability and clonal diversity in cancer disease is unclear. We isolated cancer cell

  14. Single cell mass cytometry reveals remodeling of human T cell phenotypes by varicella zoster virus.

    Science.gov (United States)

    Sen, Nandini; Mukherjee, Gourab; Arvin, Ann M

    2015-11-15

    The recent application of mass cytometry (CyTOF) to biology provides a 'systems' approach to monitor concurrent changes in multiple host cell factors at the single cell level. We used CyTOF to evaluate T cells infected with varicella zoster virus (VZV) infection, documenting virus-mediated phenotypic and functional changes caused by this T cell tropic human herpesvirus. Here we summarize our findings using two complementary panels of antibodies against surface and intracellular signaling proteins to elucidate the consequences of VZV-mediated perturbations on the surface and in signaling networks of infected T cells. CyTOF data was analyzed by several statistical, analytical and visualization tools including hierarchical clustering, orthogonal scaling, SPADE, viSNE, and SLIDE. Data from the mass cytometry studies demonstrated that VZV infection led to 'remodeling' of the surface architecture of T cells, promoting skin trafficking phenotypes and associated with concomitant activation of T-cell receptor and PI3-kinase pathways. This method offers a novel approach for understanding viral interactions with differentiated host cells important for pathogenesis. PMID:26213183

  15. Proteomic analysis of imatinib-resistant CML-T1 cells reveals calcium homeostasis as a potential therapeutic target.

    Science.gov (United States)

    Toman, O; Kabickova, T; Vit, O; Fiser, R; Polakova, K Machova; Zach, J; Linhartova, J; Vyoral, D; Petrak, J

    2016-09-01

    Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib‑resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients. PMID:27430982

  16. Cell Selection Game for Densely-Deployed Sensor and Mobile Devices In 5G Networks Integrating Heterogeneous Cells and the Internet of Things

    Science.gov (United States)

    Wang, Lusheng; Wang, Yamei; Ding, Zhizhong; Wang, Xiumin

    2015-01-01

    With the rapid development of wireless networking technologies, the Internet of Things and heterogeneous cellular networks (HCNs) tend to be integrated to form a promising wireless network paradigm for 5G. Hyper-dense sensor and mobile devices will be deployed under the coverage of heterogeneous cells, so that each of them could freely select any available cell covering it and compete for resource with others selecting the same cell, forming a cell selection (CS) game between these devices. Since different types of cells usually share the same portion of the spectrum, devices selecting overlapped cells can experience severe inter-cell interference (ICI). In this article, we study the CS game among a large amount of densely-deployed sensor and mobile devices for their uplink transmissions in a two-tier HCN. ICI is embedded with the traditional congestion game (TCG), forming a congestion game with ICI (CGI) and a congestion game with capacity (CGC). For the three games above, we theoretically find the circular boundaries between the devices selecting the macrocell and those selecting the picocells, indicated by the pure strategy Nash equilibria (PSNE). Meanwhile, through a number of simulations with different picocell radii and different path loss exponents, the collapse of the PSNE impacted by severe ICI (i.e., a large number of picocell devices change their CS preferences to the macrocell) is profoundly revealed, and the collapse points are identified. PMID:26393617

  17. Cell Selection Game for Densely-Deployed Sensor and Mobile Devices In 5G Networks Integrating Heterogeneous Cells and the Internet of Things.

    Science.gov (United States)

    Wang, Lusheng; Wang, Yamei; Ding, Zhizhong; Wang, Xiumin

    2015-01-01

    With the rapid development of wireless networking technologies, the Internet of Things and heterogeneous cellular networks (HCNs) tend to be integrated to form a promising wireless network paradigm for 5G. Hyper-dense sensor and mobile devices will be deployed under the coverage of heterogeneous cells, so that each of them could freely select any available cell covering it and compete for resource with others selecting the same cell, forming a cell selection (CS) game between these devices. Since different types of cells usually share the same portion of the spectrum, devices selecting overlapped cells can experience severe inter-cell interference (ICI). In this article, we study the CS game among a large amount of densely-deployed sensor and mobile devices for their uplink transmissions in a two-tier HCN. ICI is embedded with the traditional congestion game (TCG), forming a congestion game with ICI (CGI) and a congestion game with capacity (CGC). For the three games above, we theoretically find the circular boundaries between the devices selecting the macrocell and those selecting the picocells, indicated by the pure strategy Nash equilibria (PSNE). Meanwhile, through a number of simulations with different picocell radii and different path loss exponents, the collapse of the PSNE impacted by severe ICI (i.e., a large number of picocell devices change their CS preferences to the macrocell) is profoundly revealed, and the collapse points are identified. PMID:26393617

  18. Cell Selection Game for Densely-Deployed Sensor and Mobile Devices In 5G Networks Integrating Heterogeneous Cells and the Internet of Things

    Directory of Open Access Journals (Sweden)

    Lusheng Wang

    2015-09-01

    Full Text Available With the rapid development of wireless networking technologies, the Internet of Things and heterogeneous cellular networks (HCNs tend to be integrated to form a promising wireless network paradigm for 5G. Hyper-dense sensor and mobile devices will be deployed under the coverage of heterogeneous cells, so that each of them could freely select any available cell covering it and compete for resource with others selecting the same cell, forming a cell selection (CS game between these devices. Since different types of cells usually share the same portion of the spectrum, devices selecting overlapped cells can experience severe inter-cell interference (ICI. In this article, we study the CS game among a large amount of densely-deployed sensor and mobile devices for their uplink transmissions in a two-tier HCN. ICI is embedded with the traditional congestion game (TCG, forming a congestion game with ICI (CGI and a congestion game with capacity (CGC. For the three games above, we theoretically find the circular boundaries between the devices selecting the macrocell and those selecting the picocells, indicated by the pure strategy Nash equilibria (PSNE. Meanwhile, through a number of simulations with different picocell radii and different path loss exponents, the collapse of the PSNE impacted by severe ICI (i.e., a large number of picocell devices change their CS preferences to the macrocell is profoundly revealed, and the collapse points are identified.

  19. Elimination of self-reactive T cells in the thymus: a timeline for negative selection.

    Directory of Open Access Journals (Sweden)

    Ivan Lilyanov Dzhagalov

    Full Text Available The elimination of autoreactive T cells occurs via thymocyte apoptosis and removal by thymic phagocytes, but the sequence of events in vivo, and the relationship between thymocyte death and phagocytic clearance, are unknown. Here we address these questions by following a synchronized cohort of thymocytes undergoing negative selection within a three-dimensional thymic tissue environment, from the initial encounter with a negative selecting ligand to thymocyte death and clearance. Encounter with cognate peptide-MHC complexes results in rapid calcium flux and migratory arrest in auto-reactive thymocytes over a broad range of peptide concentrations, followed by a lag period in which gene expression changes occurred, but there was little sign of thymocyte death. Caspase 3 activation and thymocyte loss were first detectable at 2 and 3 hours, respectively, and entry of individual thymocytes into the death program occurred asynchronously over the next 10 hours. Two-photon time-lapse imaging revealed that thymocyte death and phagocytosis occurred simultaneously, often with thymocytes engulfed prior to changes in chromatin and membrane permeability. Our data provide a timeline for negative selection and reveal close coupling between cell death and clearance in the thymus.

  20. Single-Cell Analyses of ESCs Reveal Alternative Pluripotent Cell States and Molecular Mechanisms that Control Self-Renewal

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    Dmitri Papatsenko

    2015-08-01

    Full Text Available Analyses of gene expression in single mouse embryonic stem cells (mESCs cultured in serum and LIF revealed the presence of two distinct cell subpopulations with individual gene expression signatures. Comparisons with published data revealed that cells in the first subpopulation are phenotypically similar to cells isolated from the inner cell mass (ICM. In contrast, cells in the second subpopulation appear to be more mature. Pluripotency Gene Regulatory Network (PGRN reconstruction based on single-cell data and published data suggested antagonistic roles for Oct4 and Nanog in the maintenance of pluripotency states. Integrated analyses of published genomic binding (ChIP data strongly supported this observation. Certain target genes alternatively regulated by OCT4 and NANOG, such as Sall4 and Zscan10, feed back into the top hierarchical regulator Oct4. Analyses of such incoherent feedforward loops with feedback (iFFL-FB suggest a dynamic model for the maintenance of mESC pluripotency and self-renewal.

  1. An integrated cell purification and genomics strategy reveals multiple regulators of pancreas development.

    Directory of Open Access Journals (Sweden)

    Cecil M Benitez

    2014-10-01

    Full Text Available The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus.

  2. Genetically Induced Cell Death in Bulge Stem Cells Reveals Their Redundancy for Hair and Epidermal Regeneration

    OpenAIRE

    Driskell, Iwona; Oeztuerk-Winder, Feride; Humphreys, Peter; Frye, Michaela

    2014-01-01

    Adult mammalian epidermis contains multiple stem cell populations in which quiescent and more proliferative stem and progenitor populations coexist. However, the precise interrelation of these populations in homeostasis remains unclear. Here, we blocked the contribution of quiescent keratin 19 (K19)-expressing bulge stem cells to hair follicle formation through genetic ablation of the essential histone methyltransferase Setd8 that is required for the maintenance of adult skin. Deletion of Set...

  3. Phenotype-based cell-specific metabolic modeling reveals metabolic liabilities of cancer.

    Science.gov (United States)

    Yizhak, Keren; Gaude, Edoardo; Le Dévédec, Sylvia; Waldman, Yedael Y; Stein, Gideon Y; van de Water, Bob; Frezza, Christian; Ruppin, Eytan

    2014-01-01

    Utilizing molecular data to derive functional physiological models tailored for specific cancer cells can facilitate the use of individually tailored therapies. To this end we present an approach termed PRIME for generating cell-specific genome-scale metabolic models (GSMMs) based on molecular and phenotypic data. We build >280 models of normal and cancer cell-lines that successfully predict metabolic phenotypes in an individual manner. We utilize this set of cell-specific models to predict drug targets that selectively inhibit cancerous but not normal cell proliferation. The top predicted target, MLYCD, is experimentally validated and the metabolic effects of MLYCD depletion investigated. Furthermore, we tested cell-specific predicted responses to the inhibition of metabolic enzymes, and successfully inferred the prognosis of cancer patients based on their PRIME-derived individual GSMMs. These results lay a computational basis and a counterpart experimental proof of concept for future personalized metabolic modeling applications, enhancing the search for novel selective anticancer therapies. PMID:25415239

  4. Neuron-like differentiation and selective ablation of undifferentiated embryonic stem cells containing suicide gene with Oct-4 promoter.

    Science.gov (United States)

    Hara, Akira; Aoki, Hitomi; Taguchi, Ayako; Niwa, Masayuki; Yamada, Yasuhiro; Kunisada, Takahiro; Mori, Hideki

    2008-08-01

    In vivo transplantation of undifferentiated embryonic stem (ES) cells can produce teratomas with uncontrolled cell proliferation. Although ES cells may be attractive candidates for human cell-replacement therapy in the future, the major limitation of its application to the therapy is teratoma formation. In the present study, ES cells containing herpes simplex virus-thymidine kinase (HSV-tk) transgene for a suicide gene expression under the control of the Oct-4 promoter was used for ablation of undifferentiated ES cells, which may produce teratomas, using three-dimensional cell culture system allowing a multilayer cell construct. Selective ablation of undifferentiated ES cells expressing HSV-tk gene under the control of Oct-4 promoter was achieved by ganciclovir treatment. Surviving ES cells after ganciclovir treatment expressed several neuron-associated markers such as synaptophysin, beta-tubulin, vesicular glutamate transporter 1, syntaxin, protein kinase C and glial fibrillary acidic protein (GFAP) but not Oct-4. Coexpression of synaptophysin as a marker of neuronal synapse and GFAP as that of glial fibers in the surviving ES cells revealed finely structured neuronal network. Furthermore, decrease of Ki-67 proliferative index was detected in the surviving ES cells. In conclusion, selective ablation of undifferentiated ES cells by a suicide gene decreases proliferative activity and induces neuron-like differentiation in ES cells. PMID:18393636

  5. In vivo fluorescence imaging reveals the promotion of mammary tumorigenesis by mesenchymal stromal cells.

    Directory of Open Access Journals (Sweden)

    Chien-Chih Ke

    Full Text Available Mesenchymal stromal cells (MSCs are multipotent adult stem cells which are recruited to the tumor microenvironment (TME and influence tumor progression through multiple mechanisms. In this study, we examined the effects of MSCs on the tunmorigenic capacity of 4T1 murine mammary cancer cells. It was found that MSC-conditioned medium increased the proliferation, migration, and efficiency of mammosphere formation of 4T1 cells in vitro. When co-injected with MSCs into the mouse mammary fat pad, 4T1 cells showed enhanced tumor growth and generated increased spontaneous lung metastasis. Using in vivo fluorescence color-coded imaging, the interaction between GFP-expressing MSCs and RFP-expressing 4T1 cells was monitored. As few as five 4T1 cells could give rise to tumor formation when co-injected with MSCs into the mouse mammary fat pad, but no tumor was formed when five or ten 4T1 cells were implanted alone. The elevation of tumorigenic potential was further supported by gene expression analysis, which showed that when 4T1 cells were in contact with MSCs, several oncogenes, cancer markers, and tumor promoters were upregulated. Moreover, in vivo longitudinal fluorescence imaging of tumorigenesis revealed that MSCs created a vascularized environment which enhances the ability of 4T1 cells to colonize and proliferate. In conclusion, this study demonstrates that the promotion of mammary cancer progression by MSCs was achieved through the generation of a cancer-enhancing microenvironment to increase tumorigenic potential. These findings also suggest the potential risk of enhancing tumor progression in clinical cell therapy using MSCs. Attention has to be paid to patients with high risk of breast cancer when considering cell therapy with MSCs.

  6. Galactosylated poly(ethyleneglycol)-lithocholic Acid selectively kills hepatoma cells, while sparing normal liver cells.

    Science.gov (United States)

    Gankhuyag, Nomundelger; Singh, Bijay; Maharjan, Sushila; Choi, Yun-Jaie; Cho, Chong-Su; Cho, Myung-Haing

    2015-06-01

    Delivering drugs selectively to cancer cells but not to nearby normal cells is a major obstacle in drug therapy. In this study, lithocholic acid (LCA), a potent anti-cancer drug, is converted to two forms of poly(ethyleneglycol) (PEG) conjugates, viz., PEG-LCA (PL) and lactobionic acid (LBA) conjugated PEG-LCA (LPL). The latter form contains a galactose ligand in LBA to target the hepatocytes. Both forms are self-assembled to form nanoparticle formulation, and they have high potency than LCA to kill HepG2 cancer cells, sparing normal LO2 cells. Besides, LPL has high specificity to mouse liver cells in vivo. Western blot results confirm that the cell death is occurred through apoptosis induced by LPL nanoparticles. In conclusion, the induction of apoptosis and cell death is much more efficient with LPL nanoparticles than LCA molecules. PMID:25657071

  7. Age-Dependent Pancreatic Gene Regulation Reveals Mechanisms Governing Human β Cell Function.

    Science.gov (United States)

    Arda, H Efsun; Li, Lingyu; Tsai, Jennifer; Torre, Eduardo A; Rosli, Yenny; Peiris, Heshan; Spitale, Robert C; Dai, Chunhua; Gu, Xueying; Qu, Kun; Wang, Pei; Wang, Jing; Grompe, Markus; Scharfmann, Raphael; Snyder, Michael S; Bottino, Rita; Powers, Alvin C; Chang, Howard Y; Kim, Seung K

    2016-05-10

    Intensive efforts are focused on identifying regulators of human pancreatic islet cell growth and maturation to accelerate development of therapies for diabetes. After birth, islet cell growth and function are dynamically regulated; however, establishing these age-dependent changes in humans has been challenging. Here, we describe a multimodal strategy for isolating pancreatic endocrine and exocrine cells from children and adults to identify age-dependent gene expression and chromatin changes on a genomic scale. These profiles revealed distinct proliferative and functional states of islet α cells or β cells and histone modifications underlying age-dependent gene expression changes. Expression of SIX2 and SIX3, transcription factors without prior known functions in the pancreas and linked to fasting hyperglycemia risk, increased with age specifically in human islet β cells. SIX2 and SIX3 were sufficient to enhance insulin content or secretion in immature β cells. Our work provides a unique resource to study human-specific regulators of islet cell maturation and function. PMID:27133132

  8. Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging

    Science.gov (United States)

    Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

    2015-03-01

    Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction.

  9. Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres.

    Science.gov (United States)

    Schmidt, Jens C; Zaug, Arthur J; Cech, Thomas R

    2016-08-25

    Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells. We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association. Both the transient and stable association events depend on the direct interaction of the telomerase protein TERT with the telomeric protein TPP1. Our results reveal that telomerase recruitment to telomeres is driven by dynamic interactions between the rapidly diffusing telomerase and the chromosome end. PMID:27523609

  10. Wood-fired fuel cells in selected buildings

    Science.gov (United States)

    McIlveen-Wright, D. R.; McMullan, J. T.; Guiney, D. J.

    of selected buildings in rural areas, with regard to the high cost of importing other fuel, and/or lack of grid electricity, could still make these systems attractive options. Any economic analysis of these systems is beset with severe difficulties. Capital costs of the major system components are not known with any great precision. However, a guideline assessment of the payback period for such CHP systems was made. When the best available capital costs for system components were used, most of these systems were found to have unacceptably long payback periods, particularly where the fuel cell lifetimes are short, but the larger systems show the potential for a reasonable economic return.

  11. Controlled surface morphology and hydrophilicity of polycaprolactone toward selective differentiation of mesenchymal stem cells to neural like cells.

    Science.gov (United States)

    Jahani, Hoda; Jalilian, Farid Azizi; Wu, Chia-Yu; Kaviani, Saeid; Soleimani, Masoud; Abassi, Naghmeh; Ou, Keng-Liang; Hosseinkhani, Hossein

    2015-05-01

    Differentiation of mesenchymal stem cells (MSCs) into neuron cells has great potential in therapy of damaged nerve tissue. It has been shown that three-dimensional biomaterials have great ability to up regulate the expression of neuronal proteins. In this study, O2 plasma technology was used to enhance hydrophilicity of poly (ε-caprolactone) (PCL) toward selective differentiation of MSCs into neural cells. Random and aligned PCL nanofibers scaffolds were fabricated by electrospinning method and their physicochemical and mechanical properties were carried out by scanning electron microscope (SEM), contact angle, and tensile measurements. Contact angle studies of PCL and plasma treated PCL (p-PCL) nanofibers revealed significant change on the surface properties PCL nanofibers from the view point of hydrophilicity. Physiochemical studies revealed that p-PCL nanofibers were extremely hydrophilic compared with untreated PCL nanofibers which were highly hydrophobic and nonabsorbent to water. Differentiation of MSCs were carried out by inducing growth factors including basic fibroblast growth factor, nerve growth factor, and brain derived growth factor, NT3, 3-isobutyl-1-methylxanthine (IBMX) in Dulbecco's modified Eagle's medium/F12 media. Differentiated MSCs on nanofibrous scaffold were examined by immunofluorescence assay and was found to express the neuronal proteins; β-tubulin III and Map2, on day 15 after cell culture. The real-time polymerase chain reaction (RT-PCR) analysis showed that p-PCL nanofibrous scaffold could upregulate expression of Map-2 and downregulate expression of Nestin genes in nerve cells differentiated from MSCs. This study indicates that mesenchymal stem cell cultured on nanofibrous scaffold have potential differentiation to neuronal cells on and could apply in nerve tissue repair. PMID:25203786

  12. Effect of different agents onto multidrug resistant cells revealed by fluorescence correlation spectroscopy

    Science.gov (United States)

    Boutin, C.; Roche, Y.; Jaffiol, R.; Millot, J.-M.; Millot, C.; Plain, J.; Deturche, R.; Jeannesson, P.; Manfait, M.; Royer, P.

    Fluorescence correlation spectroscopy (FCS), which is a sensitive and non invasive technique, has been used to characterize the plasma membrane fluidity and heterogeneity of multidrug resistant living cells. At the single cell level, the effects of different membrane agents present in the extra-cellular medium have been analyzed. Firstly, we reveal a modification of plasma membrane microviscosity according to the addition of a fluidity modulator, benzyl alcohol. In the other hand, revertant such as verapamil and cyclosporin-A appears to act more specifically on the slow diffusion sites as microdomains.

  13. Single-cell transcriptome analysis reveals coordinated ectopic gene expression patterns in medullary thymic epithelial cells

    Science.gov (United States)

    Brennecke, Philip; Reyes, Alejandro; Pinto, Sheena; Rattay, Kristin; Nguyen, Michelle; Küchler, Rita; Huber, Wolfgang; Kyewski, Bruno; Steinmetz, Lars M.

    2015-01-01

    Expression of tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for self-tolerance induction and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA-sequencing and provide evidence for numerous recurring TRA co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process, which might involve local re-modeling of chromatin and thus ensures a comprehensive representation of the immunological self. PMID:26237553

  14. Aqueous Extracts of Selected Potentilla Species Modulate Biological Activity of Human Normal Colon Cells.

    Science.gov (United States)

    Paduch, Roman; Wiater, Adrian; Locatelli, Marcello; Pleszczyńska, Malgorzata; Tomczyk, Michal

    2015-01-01

    Potentilla L. (Rosaceae) species have been used in traditional and in folk medicine for many years. This study characterized the activity of extracts from aerial parts of selected Potentilla species: P. argentea, P. anserina, P. grandiflora and P. erecta as well as one species of closely related to the genus Potentilla, Drymocallis rupestris (syn. P. rupestris). The biological activities were analyzed using MTT, NR and DPPH assays on CCD 841 CoTr and CCD-18Co cells. Moreover, cell morphology and cytoskeletal actin F-filaments organization and IL-6 and IL-10 levels by ELISA were analyzed after 24 h of incubation. Potentilla extracts at dose levels between 25 and 250 µg/mL were analyzed. For ELISA, 15 µg/mL and 30 μg/mL were chosen. When mitochondrial succinyl dehydrogenase activity was tested (MTT assay) only extract obtained from P. erecta at lower concentrations (up to 125 µg/mL) suppressed metabolism of myofibroblasts, while epithelial cells mitochondrial enzyme activity increased after incubation with all extracts. In Neutral Red (NR) method cellular membrane disturbance of both cell cultures was found after D. rupestris and P. grandiflora addition. Moreover, strong influence on epithelial cells was also found for P. anserina. All extracts showed similar, concentration-dependent free radical scavenging (DPPH) effect. Potentilla extracts, especially at lower concentration, decreased IL-6 production in myofibroblasts but the level of the cytokine was found to be stable in epithelial cells. IL-10 analysis revealed that P. argentea, D. rupestris, P. erecta extracts decrease cytokine level in myofibroblasts, while only when higher concentration were applied, decreased cytokine level produced by epithelial cells was found. F-actin filaments staining revealed that Potentilla extracts significantly influence on cellular cytoskeleton organization. Potentilla extracts influence on cells of human colon wall lining modulating the main features of them (viability

  15. Synergy analysis reveals association between insulin signaling and desmoplakin expression in palmitate treated HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Xuewei Wang

    Full Text Available The regulation of complex cellular activities in palmitate treated HepG2 cells, and the ensuing cytotoxic phenotype, involves cooperative interactions between genes. While previous approaches have largely focused on identifying individual target genes, elucidating interacting genes has thus far remained elusive. We applied the concept of information synergy to reconstruct a "gene-cooperativity" network for palmititate-induced cytotoxicity in liver cells. Our approach integrated gene expression data with metabolic profiles to select a subset of genes for network reconstruction. Subsequent analysis of the network revealed insulin signaling as the most significantly enriched pathway, and desmoplakin (DSP as its top neighbor. We determined that palmitate significantly reduces DSP expression, and treatment with insulin restores the lost expression of DSP. Insulin resistance is a common pathological feature of fatty liver and related ailments, whereas loss of DSP has been noted in liver carcinoma. Reduced DSP expression can lead to loss of cell-cell adhesion via desmosomes, and disrupt the keratin intermediate filament network. Our findings suggest that DSP expression may be perturbed by palmitate and, along with insulin resistance, may play a role in palmitate induced cytotoxicity, and serve as potential targets for further studies on non-alcoholic fatty liver disease (NAFLD.

  16. Mitochondrial DNA mutations in renal cell carcinomas revealed no general impact on energy metabolism

    OpenAIRE

    Meierhofer, D.; Mayr, J. A.; Fink, K.; Schmeller, N.; Kofler, B; Sperl, W.

    2006-01-01

    Previously, renal cell carcinoma tissues were reported to display a marked reduction of components of the respiratory chain. To elucidate a possible relationship between tumourigenesis and alterations of oxidative phosphorylation, we screened for mutations of the mitochondrial DNA (mtDNA) in renal carcinoma tissues and patient-matched normal kidney cortex. Seven of the 15 samples investigated revealed at least one somatic heteroplasmic mutation as determined by denaturating HPLC analysis (DHP...

  17. Functional Features of Trans-differentiated Hair Cells Mediated by Atoh1 Reveals a Primordial Mechanism

    OpenAIRE

    Yang, Juanmei; Bouvron, Sonia; Lv, Ping; Chi, Fanglu; Yamoah, Ebenezer N.

    2012-01-01

    Evolution has transformed a simple ear with few vestibular maculae into a complex 3-dimensional structure consisting of nine distinct endorgans. It is debatable whether the sensory epithelia underwent progressive segregation or emerged from distinct sensory patches. To address these uncertainties we examined the morphological and functional phenotype of trans-differentiated rat hair cells to reveal their primitive or endorgan-specific origins. Additionally, it is uncertain how Atoh1-mediated ...

  18. Early events leading to erythroid differentiation in mouse Friend cells revealed by cell fusion experiments

    International Nuclear Information System (INIS)

    Cell fusion with two genetically marked Friend (murine erythroleukemia) cells has made it possible to characterize the very early events leading to erythroid differentiation, particularly the nature of reactions initiated by inducers such as dimethyl sulfoxide. We have found that brief exposure of Friend cells to dimethyl sulfoxide (as well as butyric acid or hexamethylene-bisacetamide) induces an early cellular activity required for erythroid differentiation which is detected only by fusion with ultraviolet-irradiated cells. The induction process of this activity consists of at least two distinct stages. In the first stage, the reaction proceeds without supply of metabolites from the medium and exhibits sensitivity to tumor promoters. The second stage is tightly coupled to cellular metabolic activity, notably protein synthesis. Under normal conditions, the induced activity is short-lived, suggesting turnover of the molecules responsible for this activity. There appears to be a signal produced following dimethyl sulfoxide pulse which acts as an inducer for this activity. The signal remains active for as long as 40 hr when protein synthesis is blocked

  19. Quantitative proteomics reveals middle infrared radiation-interfered networks in breast cancer cells.

    Science.gov (United States)

    Chang, Hsin-Yi; Li, Ming-Hua; Huang, Tsui-Chin; Hsu, Chia-Lang; Tsai, Shang-Ru; Lee, Si-Chen; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2015-02-01

    Breast cancer is one of the leading cancer-related causes of death worldwide. Treatment of triple-negative breast cancer (TNBC) is complex and challenging, especially when metastasis has developed. In this study, we applied infrared radiation as an alternative approach for the treatment of TNBC. We used middle infrared (MIR) with a wavelength range of 3-5 μm to irradiate breast cancer cells. MIR significantly inhibited cell proliferation in several breast cancer cells but did not affect the growth of normal breast epithelial cells. We performed iTRAQ-coupled LC-MS/MS analysis to investigate the MIR-triggered molecular mechanisms in breast cancer cells. A total of 1749 proteins were identified, quantified, and subjected to functional enrichment analysis. From the constructed functionally enriched network, we confirmed that MIR caused G2/M cell cycle arrest, remodeled the microtubule network to an astral pole arrangement, altered the actin filament formation and focal adhesion molecule localization, and reduced cell migration activity and invasion ability. Our results reveal the coordinative effects of MIR-regulated physiological responses in concentrated networks, demonstrating the potential implementation of infrared radiation in breast cancer therapy. PMID:25556991

  20. Stem cell-like differentiation potentials of endometrial side population cells as revealed by a newly developed in vivo endometrial stem cell assay.

    Directory of Open Access Journals (Sweden)

    Kaoru Miyazaki

    Full Text Available BACKGROUND: Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP, but not endometrial main population cells (EMP, exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. METHODOLOGY/PRINCIPAL FINDINGS: ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom, a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. CONCLUSIONS/SIGNIFICANCE: We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo

  1. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

    Directory of Open Access Journals (Sweden)

    Ingrid R. Cordeiro

    2015-09-01

    Full Text Available Human adipose-derived stromal cells (hADSC are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1 regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

  2. Neisseria lactamica selectively induces mitogenic proliferation of the naive B cell pool via cell surface Ig.

    Science.gov (United States)

    Vaughan, Andrew T; Brackenbury, Louise S; Massari, Paola; Davenport, Victoria; Gorringe, Andrew; Heyderman, Robert S; Williams, Neil A

    2010-09-15

    Neisseria lactamica is a commensal bacteria that colonizes the human upper respiratory tract mucosa during early childhood. In contrast to the closely related opportunistic pathogen Neisseria meningitidis, there is an absence of adaptive cell-mediated immunity to N. lactamica during the peak age of carriage. Instead, outer membrane vesicles derived from N. lactamica mediate a B cell-dependent proliferative response in mucosal mononuclear cells that is associated with the production of polyclonal IgM. We demonstrate in this study that this is a mitogenic human B cell response that occurs independently of T cell help and any other accessory cell population. The ability to drive B cell proliferation is a highly conserved property and is present in N. lactamica strains derived from diverse clonal complexes. CFSE staining of purified human tonsillar B cells demonstrated that naive IgD(+) and CD27(-) B cells are selectively induced to proliferate by outer membrane vesicles, including the innate CD5(+) subset. Neither purified lipooligosaccharide nor PorB from N. lactamica is likely to be responsible for this activity. Prior treatment of B cells with pronase to remove cell-surface Ig or treatment with BCR-specific Abs abrogated the proliferative response to N. lactamica outer membrane vesicles, suggesting that this mitogenic response is dependent upon the BCR. PMID:20709949

  3. A genome-wide systematic analysis reveals different and predictive proliferation expression signatures of cancerous vs. non-cancerous cells.

    Directory of Open Access Journals (Sweden)

    Yedael Y Waldman

    Full Text Available Understanding cell proliferation mechanisms has been a long-lasting goal of the scientific community and specifically of cancer researchers. Previous genome-scale studies of cancer proliferation determinants have mainly relied on knockdown screens aimed to gauge their effects on cancer growth. This powerful approach has several limitations such as off-target effects, partial knockdown, and masking effects due to functional backups. Here we employ a complementary approach and assign each gene a cancer Proliferation Index (cPI that quantifies the association between its expression levels and growth rate measurements across 60 cancer cell lines. Reassuringly, genes found essential in cancer gene knockdown screens exhibit significant positive cPI values, while tumor suppressors exhibit significant negative cPI values. Cell cycle, DNA replication, splicing and protein production related processes are positively associated with cancer proliferation, while cellular migration is negatively associated with it - in accordance with the well known "go or grow" dichotomy. A parallel analysis of genes' non-cancerous proliferation indices (nPI across 224 lymphoblastoid cell lines reveals surprisingly marked differences between cancerous and non-cancerous proliferation. These differences highlight genes in the translation and spliceosome machineries as selective cancer proliferation-associated proteins. A cross species comparison reveals that cancer proliferation resembles that of microorganisms while non-cancerous proliferation does not. Furthermore, combining cancerous and non-cancerous proliferation signatures leads to enhanced prediction of patient outcome and gene essentiality in cancer. Overall, these results point to an inherent difference between cancerous and non-cancerous proliferation determinants, whose understanding may contribute to the future development of novel cancer-specific anti-proliferative drugs.

  4. Metabolomic profiles reveal key metabolic changes in heat stress-treated mouse Sertoli cells.

    Science.gov (United States)

    Xu, Bo; Chen, Minjian; Ji, Xiaoli; Yao, Mengmeng; Mao, Zhilei; Zhou, Kun; Xia, Yankai; Han, Xiao; Tang, Wei

    2015-10-01

    Heat stress (HS) is a potential harmful factor for male reproduction. However, the effect of HS on Sertoli cells is largely unknown. In this study, the metabolic changes in Sertoli cell line were analyzed after HS treatment. Metabolomic analysis revealed that carnitine, 2-hydroxy palmitic acid, nicotinic acid, niacinamide, adenosine monophosphate, glutamine and creatine were the key changed metabolites. We found the expression levels of BTB factors (Connexin43, ZO-1, Vimentin, Claudin1, Claudin5) were disrupted in TM-4 cells after HS treatment, which were recovered by the addition of carnitine. RT-PCR indicated that the mRNA levels of inflammatory cytokines (IL-1α, IL-1β, IL-6) were increased after HS treatment, and their related miRNAs (miR-132, miR-431, miR-543) levels were decreased. Our metabolomic data provided a novel understanding of metabolic changes in male reproductive cells after HS treatment and revealed that HS-induced changes of BTB factors and inflammatory status might be caused by the decreased carnitine after HS treatment. PMID:26165742

  5. The role of negative selection in protein evolution revealed through the energetics of the native state ensemble.

    Science.gov (United States)

    Hoffmann, Jordan; Wrabl, James O; Hilser, Vincent J

    2016-04-01

    Knowing the determinants of conformational specificity is essential for understanding protein structure, stability, and fold evolution. To address this issue, a novel statistical measure of energetic compatibility between sequence and structure was developed using an experimentally validated model of the energetics of the native state ensemble. This approach successfully matched sequences from a diverse subset of the human proteome to their respective folds. Unexpectedly, significant energetic compatibility between ostensibly unrelated sequences and structures was also observed. Interrogation of these matches revealed a general framework for understanding the origins of conformational specificity within a proteome: specificity is a complex function of both the ability of a sequence to adopt folds other than the native, and ability of a fold to accommodate sequences other than the native. The regional variation in energetic compatibility indicates that the compatibility is dominated by incompatibility of sequence for alternative fold segments, suggesting that evolution of protein sequences has involved substantial negative selection, with certain segments serving as "gatekeepers" that presumably prevent alternative structures. Beyond these global trends, a size dependence exists in the degree to which the energetic compatibility is determined from negative selection, with smaller proteins displaying more negative selection. This partially explains how short sequences can adopt unique folds, despite the higher probability in shorter proteins for small numbers of mutations to increase compatibility with other folds. In providing evolutionary ground rules for the thermodynamic relationship between sequence and fold, this framework imparts valuable insight for rational design of unique folds or fold switches. Proteins 2016; 84:435-447. © 2016 Wiley Periodicals, Inc. PMID:26800099

  6. Modeling chronic myeloid leukemia in immunodeficient mice reveals expansion of aberrant mast cells and accumulation of pre-B cells

    International Nuclear Information System (INIS)

    Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that, if not treated, will progress into blast crisis (BC) of either myeloid or B lymphoid phenotype. The BCR-ABL1 fusion gene, encoding a constitutively active tyrosine kinase, is thought to be sufficient to cause chronic phase (CP) CML, whereas additional genetic lesions are needed for progression into CML BC. To generate a humanized CML model, we retrovirally expressed BCR-ABL1 in the cord blood CD34+ cells and transplanted these into NOD-SCID (non-obese diabetic/severe-combined immunodeficient) interleukin-2-receptor γ-deficient mice. In primary mice, BCR-ABL1 expression induced an inflammatory-like state in the bone marrow and spleen, and mast cells were the only myeloid lineage specifically expanded by BCR-ABL1. Upon secondary transplantation, the pronounced inflammatory phenotype was lost and mainly human mast cells and macrophages were found in the bone marrow. Moreover, a striking block at the pre-B-cell stage was observed in primary mice, resulting in an accumulation of pre-B cells. A similar block in B-cell differentiation could be confirmed in primary cells from CML patients. Hence, this humanized mouse model of CML reveals previously unexplored features of CP CML and should be useful for further studies to understand the disease pathogenesis of CML

  7. Detection of an Immunogenic HERV-E Envelope with Selective Expression in Clear Cell Kidney Cancer.

    Science.gov (United States)

    Cherkasova, Elena; Scrivani, Claire; Doh, Susan; Weisman, Quinn; Takahashi, Yoshiyuki; Harashima, Nanae; Yokoyama, Hisayuki; Srinivasan, Ramaprasad; Linehan, W Marston; Lerman, Michael I; Childs, Richard W

    2016-04-15

    VHL-deficient clear cell renal cell carcinomas (ccRCC), the most common form of kidney cancer, express transcripts derived from the novel human endogenous retrovirus HERV-E (named CT-RCC HERV-E). In this study, we define a transcript encoding the entire envelope gene of HERV-E as expressed selectively in ccRCC tumors, as distinct from normal kidney tissues or other tumor types. Sequence analysis of this envelope transcript revealed long open reading frames encoding putative surface and transmembrane envelope proteins. Retroviral envelopes are known to be capable of eliciting immunity in humans. Accordingly, we found that HLA-A*0201-restricted peptides predicted to be products of the CT-RCC HERV-E envelope transcript-stimulated CD8(+) T cells, which could recognize HLA-A*0201-positive HERV-E-expressing kidney tumor cells. Overall, our results offer evidence of unique HERV-E envelope peptides presented on the surface of ccRCC cells, offering potentially useful tumor-restricted targets for T-cell-based immunotherapy of kidney cancer. Cancer Res; 76(8); 2177-85. ©2016 AACR. PMID:26862115

  8. Transcriptomic changes in human renal proximal tubular cells revealed under hypoxic conditions by RNA sequencing.

    Science.gov (United States)

    Yu, Wenmin; Li, Yiping; Wang, Zhi; Liu, Lei; Liu, Jing; Ding, Fengan; Zhang, Xiaoyi; Cheng, Zhengyuan; Chen, Pingsheng; Dou, Jun

    2016-09-01

    Chronic hypoxia often occurs among patients with chronic kidney disease (CKD). Renal proximal tubular cells may be the primary target of a hypoxic insult. However, the underlying transcriptional mechanisms remain undefined. In this study, we revealed the global changes in gene expression in HK‑2 human renal proximal tubular cells under hypoxic and normoxic conditions. We analyzed the transcriptome of HK‑2 cells exposed to hypoxia for 24 h using RNA sequencing. A total of 279 differentially expressed genes was examined, as these genes could potentially explain the differences in HK‑2 cells between hypoxic and normoxic conditions. Moreover, 17 genes were validated by qPCR, and the results were highly concordant with the RNA seqencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these differentially expressed genes. The upregulated genes appeared to be significantly enriched in the pathyway of extracellular matrix (ECM)-receptor interaction, and in paticular, the pathway of renal cell carcinoma was upregulated under hypoxic conditions. The downregulated genes were enriched in the signaling pathway related to antigen processing and presentation; however, the pathway of glutathione metabolism was downregulated. Our analysis revealed numerous novel transcripts and alternative splicing events. Simultaneously, we also identified a large number of single nucleotide polymorphisms, which will be a rich resource for future marker development. On the whole, our data indicate that transcriptome analysis provides valuable information for a more in depth understanding of the molecular mechanisms in CKD and renal cell carcinoma. PMID:27432315

  9. A computer-assisted 3D model for analyzing the aggregation of tumorigenic cells reveals specialized behaviors and unique cell types that facilitate aggregate coalescence.

    Directory of Open Access Journals (Sweden)

    Amanda Scherer

    Full Text Available We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity, and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named "facilitators" and "probes." A third cell type, the "dervish", is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.

  10. Drug Treatment of Cancer Cell Lines: A Way to Select for Cancer Stem Cells?

    Energy Technology Data Exchange (ETDEWEB)

    Chiodi, Ilaria; Belgiovine, Cristina; Donà, Francesca; Scovassi, A. Ivana; Mondello, Chiara, E-mail: mondello@igm.cnr.it [Institute of Molecular Genetics, CNR, via Abbiategrasso 207, 27100 Pavia (Italy)

    2011-03-04

    Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

  11. Drug Treatment of Cancer Cell Lines: A Way to Select for Cancer Stem Cells?

    International Nuclear Information System (INIS)

    Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs

  12. Pyramidal cells in prefrontal cortex: comparative observations reveal unparalleled specializations in neuronal structure among primate species.

    Directory of Open Access Journals (Sweden)

    Guy eElston

    2011-02-01

    Full Text Available The most ubiquitous neuron in the cerebral cortex, the pyramidal cell, is characterised by markedly different dendritic structure among different cortical areas. The complex pyramidal cell phenotype in granular prefrontal cortex (gPFC of higher primates endows specific biophysical properties and patterns of connectivity, which differ to those in other cortical regions. However, within the gPFC, data have been sampled from only a select few cortical areas. The gPFC of species such as human and macaque monkey includes more than 10 cortical areas. It remains unknown as to what degree pyramidal cell structure may vary among these cortical areas. Here we undertook a survey of pyramidal cells in the dorsolateral, medial and orbital gPFC of cercopethicid primates. We found marked heterogeneity in pyramidal cell structure within and between these regions. Moreover, trends for gradients in neuronal complexity varied among species. As neuron structure determines it’s computational abilities and memory storage capacity and connectivity, we propose that these specializations in the pyramidal cell phenotype are an important determinant of species specific executive cortical functions in primates.

  13. Secretome analysis of multiple pancreatic cancer cell lines reveals perturbations of key functional networks.

    Science.gov (United States)

    Schiarea, Silvia; Solinas, Graziella; Allavena, Paola; Scigliuolo, Graziana Maria; Bagnati, Renzo; Fanelli, Roberto; Chiabrando, Chiara

    2010-09-01

    The cancer secretome is a rich repository in which to mine useful information for both cancer biology and clinical oncology. To help understand the mechanisms underlying the progression of pancreatic cancer, we characterized the secretomes of four human pancreatic ductal adenocarcinoma (PDAC) cell lines versus a normal counterpart. To this end, we used a proteomic workflow based on high-confidence protein identification by mass spectrometry, semiquantitation by a label-free approach, and network enrichment analysis by a system biology tool. Functional networks significantly enriched with PDAC-dysregulated proteins included not only expected alterations within key mechanisms known to be relevant for tumor progression (e.g., cell-cell/cell-matrix adhesion, extracellular matrix remodeling, and cytoskeleton rearrangement), but also other extensive, coordinated perturbations never observed in pancreatic cancer. In particular, we highlighted perturbations possibly favoring tumor progression through immune escape (i.e., inhibition of the complement system, deficiency of selected proteasome components within the antigen-presentation machinery, and inhibition of T cell cytoxicity), and a defective protein folding machinery. Among the proteins found concordantly oversecreted in all of our PDAC cell lines, many are reportedly overexpressed in pancreatic cancer (e.g., CD9 and Vimentin), while others (PLOD3, SH3L3, PCBP1, and SFRS1) represent novel PDAC-secreted proteins that may be worth investigating. PMID:20687567

  14. An Electrochemical Cell for Selective Lithium Capture from Seawater.

    Science.gov (United States)

    Kim, Joo-Seong; Lee, Yong-Hee; Choi, Seungyeon; Shin, Jaeho; Dinh, Hung-Cuong; Choi, Jang Wook

    2015-08-18

    Lithium (Li) is a core element of Li-ion batteries (LIBs). Recent developments in mobile electronics such as smartphones and tablet PCs as well as advent of large-scale LIB applications including electrical vehicles and grid-level energy storage systems have led to an increase in demand for LIBs, giving rise to a concern on the availability and market price of Li resources. However, the current Lime-Soda process that is responsible for greater than 80% of worldwide Li resource supply is applicable only in certain regions on earth where the Li concentrations are sufficiently high (salt lakes or salt pans). Moreover, not only is the process time-consuming (12-18 months), but post-treatments are also required for the purification of Li. Here, we have devised a location-independent electrochemical system for Li capture, which can operate within a short time period (a few hours to days). By engaging olivine LiFePO4 active electrode that improves interfacial properties via polydopamine coating, the electrochemical cell achieves 4330 times amplification in Li/Na ion selectivity (Li/Na molar ratio of initial solution = 0.01 and Li/Na molar ratio of final electrode = 43.3). In addition, the electrochemical system engages an I(-)/I3(-) redox couple in the other electrode for balancing of the redox states on both electrode sides and sustainable operations of the entire cell. Based on the electrochemical results, key material and interfacial properties that affect the selectivity in Li capture are identified. PMID:25920476

  15. Repair of Ischemic Injury by Pluripotent Stem Cell Based Cell Therapy without Teratoma through Selective Photosensitivity

    Directory of Open Access Journals (Sweden)

    Seung-Ju Cho

    2015-12-01

    Full Text Available Stem-toxic small molecules have been developed to induce selective cell death of pluripotent stem cells (PSCs to lower the risk of teratoma formation. However, despite their high efficacies, chemical-based approaches may carry unexpected toxicities on specific differentiated cell types. Herein, we took advantage of KillerRed (KR as a suicide gene, to selectively induce phototoxicity using visible light via the production of reactive oxygen species. PSCs in an undifferentiated state that exclusively expressed KR (KR-PSCs were eliminated by a single exposure to visible light. This highly selective cell death in KR-PSCs was exploited to successfully inhibit teratoma formation. In particular, endothelial cells from KR-mPSCs remained fully functional in vitro and sufficient to repair ischemic injury in vivo regardless of light exposure, suggesting that a genetic approach in which KR is expressed in a tightly controlled manner would be a viable strategy to inhibit teratoma formation for future safe PSC-based therapies.

  16. Mechanism of selective VEGF-A binding by neuropilin-1 reveals a basis for specific ligand inhibition.

    Directory of Open Access Journals (Sweden)

    Matthew W Parker

    Full Text Available Neuropilin (Nrp receptors function as essential cell surface receptors for the Vascular Endothelial Growth Factor (VEGF family of proangiogenic cytokines and the semaphorin 3 (Sema3 family of axon guidance molecules. There are two Nrp homologues, Nrp1 and Nrp2, which bind to both overlapping and distinct members of the VEGF and Sema3 family of molecules. Nrp1 specifically binds the VEGF-A(164/5 isoform, which is essential for developmental angiogenesis. We demonstrate that VEGF-A specific binding is governed by Nrp1 residues in the b1 coagulation factor domain surrounding the invariant Nrp C-terminal arginine binding pocket. Further, we show that Sema3F does not display the Nrp-specific binding to the b1 domain seen with VEGF-A. Engineered soluble Nrp receptor fragments that selectively sequester ligands from the active signaling complex are an attractive modality for selectively blocking the angiogenic and chemorepulsive functions of Nrp ligands. Utilizing the information on Nrp ligand binding specificity, we demonstrate Nrp constructs that specifically sequester Sema3 in the presence of VEGF-A. This establishes that unique mechanisms are used by Nrp receptors to mediate specific ligand binding and that these differences can be exploited to engineer soluble Nrp receptors with specificity for Sema3.

  17. Tissue-Specific Gain of RTK Signalling Uncovers Selective Cell Vulnerability during Embryogenesis.

    Directory of Open Access Journals (Sweden)

    Yannan Fan

    Full Text Available The successive events that cells experience throughout development shape their intrinsic capacity to respond and integrate RTK inputs. Cellular responses to RTKs rely on different mechanisms of regulation that establish proper levels of RTK activation, define duration of RTK action, and exert quantitative/qualitative signalling outcomes. The extent to which cells are competent to deal with fluctuations in RTK signalling is incompletely understood. Here, we employ a genetic system to enhance RTK signalling in a tissue-specific manner. The chosen RTK is the hepatocyte growth factor (HGF receptor Met, an appropriate model due to its pleiotropic requirement in distinct developmental events. Ubiquitously enhanced Met in Cre/loxP-based Rosa26(stopMet knock-in context (Del-R26(Met reveals that most tissues are capable of buffering enhanced Met-RTK signalling thus avoiding perturbation of developmental programs. Nevertheless, this ubiquitous increase of Met does compromise selected programs such as myoblast migration. Using cell-type specific Cre drivers, we genetically showed that altered myoblast migration results from ectopic Met expression in limb mesenchyme rather than in migrating myoblasts themselves. qRT-PCR analyses show that ectopic Met in limbs causes molecular changes such as downregulation in the expression levels of Notum and Syndecan4, two known regulators of morphogen gradients. Molecular and functional studies revealed that ectopic Met expression in limb mesenchyme does not alter HGF expression patterns and levels, but impairs HGF bioavailability. Together, our findings show that myoblasts, in which Met is endogenously expressed, are capable of buffering increased RTK levels, and identify mesenchymal cells as a cell type vulnerable to ectopic Met-RTK signalling. These results illustrate that embryonic cells are sensitive to alterations in the spatial distribution of RTK action, yet resilient to fluctuations in signalling levels of an

  18. Curcumin and cancer cells: how many ways can curry kill tumor cells selectively?

    Science.gov (United States)

    Ravindran, Jayaraj; Prasad, Sahdeo; Aggarwal, Bharat B

    2009-09-01

    Cancer is a hyperproliferative disorder that is usually treated by chemotherapeutic agents that are toxic not only to tumor cells but also to normal cells, so these agents produce major side effects. In addition, these agents are highly expensive and thus not affordable for most. Moreover, such agents cannot be used for cancer prevention. Traditional medicines are generally free of the deleterious side effects and usually inexpensive. Curcumin, a component of turmeric (Curcuma longa), is one such agent that is safe, affordable, and efficacious. How curcumin kills tumor cells is the focus of this review. We show that curcumin modulates growth of tumor cells through regulation of multiple cell signaling pathways including cell proliferation pathway (cyclin D1, c-myc), cell survival pathway (Bcl-2, Bcl-xL, cFLIP, XIAP, c-IAP1), caspase activation pathway (caspase-8, 3, 9), tumor suppressor pathway (p53, p21) death receptor pathway (DR4, DR5), mitochondrial pathways, and protein kinase pathway (JNK, Akt, and AMPK). How curcumin selectively kills tumor cells, and not normal cells, is also described in detail. PMID:19590964

  19. A PCNA-derived cell permeable peptide selectively inhibits neuroblastoma cell growth.

    Directory of Open Access Journals (Sweden)

    Long Gu

    Full Text Available Proliferating cell nuclear antigen (PCNA, through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA, which was significantly expressed in a broad range of cancer cells and tumor tissues, but not in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interconnector domain of PCNA that is known to be a major binding site for many of PCNA's interacting proteins. We hypothesized that therapeutic agents targeting protein-protein interactions mediated through this region may confer differential toxicity to normal and malignant cells. To test this hypothesis, we designed a cell permeable peptide containing the PCNA L126-Y133 sequence. Here, we report that this peptide selectively kills human neuroblastoma cells, especially those with MYCN gene amplification, with much less toxicity to non-malignant human cells. Mechanistically, the peptide is able to block PCNA interactions in cancer cells. It interferes with DNA synthesis and homologous recombination-mediated double-stranded DNA break repair, resulting in S-phase arrest, accumulation of DNA damage, and enhanced sensitivity to cisplatin. These results demonstrate conceptually the utility of this peptide for treating neuroblastomas, particularly, the unfavorable MYCN-amplified tumors.

  20. [Acute intestinal obstruction revealing enteropathy associated t-cell lymphoma, about a case].

    Science.gov (United States)

    Garba, Abdoul Aziz; Adamou, Harissou; Magagi, Ibrahim Amadou; Brah, Souleymane; Habou, Oumarou

    2016-01-01

    Enteropathy associated T-cell lymphoma (EATL) is a rare complication of celiac disease (CD). We report a case of EATL associated with CD revealed by acute intestinal obstruction. A North African woman of 38 years old with a history of infertility and chronic abdominal pain was admitted in emergency with acute intestinal obstruction. During the surgery, we found a tumor on the small intestine with mesenteric lymphadenopathy. Histology and immunohistochemistry of the specimen objectified a digestive T lymphoma CD3+ and immunological assessment of celiac disease was positive. The diagnosis of EATL was thus retained. Chemotherapy (CHOEP protocol) was established as well as gluten-free diet with a complete response to treatment. The EATL is a rare complication of CD that can be revealed by intestinal obstruction. The prognosis can be improved by early treatment involving surgery and chemotherapy. Its prevention requires early diagnosis of celiac and gluten-free diets. PMID:27217874

  1. Adaptability and selectivity of human peroxisome proliferator-activated receptor (PPAR) pan agonists revealed from crystal structures

    International Nuclear Information System (INIS)

    The structures of the ligand-binding domains (LBDs) of human peroxisome proliferator-activated receptors (PPARα, PPARγ and PPARδ) in complexes with a pan agonist, an α/δ dual agonist and a PPARδ-specific agonist were determined. The results explain how each ligand is recognized by the PPAR LBDs at an atomic level. Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor family, which is defined as transcriptional factors that are activated by the binding of ligands to their ligand-binding domains (LBDs). Although the three PPAR subtypes display different tissue distribution patterns and distinct pharmacological profiles, they all are essentially related to fatty-acid and glucose metabolism. Since the PPARs share similar three-dimensional structures within the LBDs, synthetic ligands which simultaneously activate two or all of the PPARs could be potent candidates in terms of drugs for the treatment of abnormal metabolic homeostasis. The structures of several PPAR LBDs were determined in complex with synthetic ligands, derivatives of 3-(4-alkoxyphenyl)propanoic acid, which exhibit unique agonistic activities. The PPARα and PPARγ LBDs were complexed with the same pan agonist, TIPP-703, which activates all three PPARs and their crystal structures were determined. The two LBD–ligand complex structures revealed how the pan agonist is adapted to the similar, but significantly different, ligand-binding pockets of the PPARs. The structures of the PPARδ LBD in complex with an α/δ-selective ligand, TIPP-401, and with a related δ-specific ligand, TIPP-204, were also determined. The comparison between the two PPARδ complexes revealed how each ligand exhibits either a ‘dual selective’ or ‘single specific’ binding mode

  2. Gene expression profiling of dendritic cells reveals important mechanisms associated with predisposition to Staphylococcus infections.

    Directory of Open Access Journals (Sweden)

    Mehdi Toufeer

    Full Text Available BACKGROUND: Staphylococcus aureus is a major pathogen of humans and animals and emerging antibiotic-resistant strains have further increased the concern of this health issue. Host genetics influence susceptibility to S. aureus infections, and the genes determining the outcome of infections should be identified to find alternative therapies to treatment with antibiotics. Here, we used outbred animals from a divergent selection based on susceptibility towards Staphylococcus infection to explore host immunogenetics. METHODOLOGY/PRINCIPAL FINDINGS: We investigated how dendritic cells respond to heat-inactivated S. aureus and whether dendritic cells from animals showing different degrees of susceptibility had distinct gene expression profiles. We measured gene expression levels of in vitro S. aureus-stimulated bone marrow-derived dendritic cells at three different time points (0, 3 and 8 hrs by using 15 k ovine Agilent microarrays. Furthermore, differential expression of a selected number of genes was confirmed by RT-qPCR. Gene signatures of stimulated DCs were obtained and showed that genes involved in the inflammatory process and T helper cell polarization were highly up-regulated upon stimulation. Moreover, a set of 204 genes were statistically differentially expressed between susceptible and resistant animals, and grouped them according to their predisposition to staphylococcal infection. Interestingly, over-expression of the C1q and Ido1 genes was observed in the resistant line and suggested a role of classical pathway of complement and early regulation of inflammation pathways, respectively. On the contrary, over expression of genes involved in the IL1R pathway was observed in susceptible animals. Furthermore, the leucocyte extravasation pathway was also found to be dominant in the susceptible line. CONCLUSION/SIGNIFICANCE: We successfully obtained Staphylococcus aureus associated gene expression of ovine BM-DC in an 8-hour kinetics experiment

  3. Gene Expression Profiling of Dendritic Cells Reveals Important Mechanisms Associated with Predisposition to Staphylococcus Infections

    Science.gov (United States)

    Toufeer, Mehdi; Bonnefont, Cécile M. D.; Foulon, Eliane; Caubet, Cécile; Tasca, Christian; Aurel, Marie-Rose; Robert-Granié, Christèle; Rupp, Rachel; Foucras, Gilles

    2011-01-01

    Background Staphylococcus aureus is a major pathogen of humans and animals and emerging antibiotic-resistant strains have further increased the concern of this health issue. Host genetics influence susceptibility to S. aureus infections, and the genes determining the outcome of infections should be identified to find alternative therapies to treatment with antibiotics. Here, we used outbred animals from a divergent selection based on susceptibility towards Staphylococcus infection to explore host immunogenetics. Methodology/Principal Findings We investigated how dendritic cells respond to heat-inactivated S. aureus and whether dendritic cells from animals showing different degrees of susceptibility had distinct gene expression profiles. We measured gene expression levels of in vitro S. aureus-stimulated bone marrow-derived dendritic cells at three different time points (0, 3 and 8 hrs) by using 15 k ovine Agilent microarrays. Furthermore, differential expression of a selected number of genes was confirmed by RT-qPCR. Gene signatures of stimulated DCs were obtained and showed that genes involved in the inflammatory process and T helper cell polarization were highly up-regulated upon stimulation. Moreover, a set of 204 genes were statistically differentially expressed between susceptible and resistant animals, and grouped them according to their predisposition to staphylococcal infection. Interestingly, over-expression of the C1q and Ido1 genes was observed in the resistant line and suggested a role of classical pathway of complement and early regulation of inflammation pathways, respectively. On the contrary, over expression of genes involved in the IL1R pathway was observed in susceptible animals. Furthermore, the leucocyte extravasation pathway was also found to be dominant in the susceptible line. Conclusion/Significance We successfully obtained Staphylococcus aureus associated gene expression of ovine BM-DC in an 8-hour kinetics experiment. The distinct

  4. Replicator Dynamics of of Cancer Stem Cell; Selection in the Presence of Differentiation and Plasticity

    OpenAIRE

    Kaveh, Kamran; Kohandel, Mohammad; Sivaloganathan, Siv

    2014-01-01

    Stem cells have the potential to produce lineages of non-stem cell populations (differentiated cells) via a ubiquitous hierarchal division scheme. Differentiation of a stem cell into (partially) differentiated cells can happen either symmetrically or asymmetrically. The selection dynamics of a mutant cancer stem cell should be investigated in the light of a stem cell proliferation hierarchy and presence of a non-stem cell population. By constructing a three-compartment Moran-type model compos...

  5. Magnesium Fluoride Electron-Selective Contacts for Crystalline Silicon Solar Cells.

    Science.gov (United States)

    Wan, Yimao; Samundsett, Chris; Bullock, James; Allen, Thomas; Hettick, Mark; Yan, Di; Zheng, Peiting; Zhang, Xinyu; Cui, Jie; McKeon, Josephine; Javey, Ali; Cuevas, Andres

    2016-06-15

    In this study, we present a novel application of thin magnesium fluoride films to form electron-selective contacts to n-type crystalline silicon (c-Si). This allows the demonstration of a 20.1%-efficient c-Si solar cell. The electron-selective contact is composed of deposited layers of amorphous silicon (∼6.5 nm), magnesium fluoride (∼1 nm), and aluminum (∼300 nm). X-ray photoelectron spectroscopy reveals a work function of 3.5 eV at the MgF2/Al interface, significantly lower than that of aluminum itself (∼4.2 eV), enabling an Ohmic contact between the aluminum electrode and n-type c-Si. The optimized contact structure exhibits a contact resistivity of ∼76 mΩ·cm(2), sufficiently low for a full-area contact to solar cells, together with a very low contact recombination current density of ∼10 fA/cm(2). We demonstrate that electrodes functionalized with thin magnesium fluoride films significantly improve the performance of silicon solar cells. The novel contacts can potentially be implemented also in organic optoelectronic devices, including photovoltaics, thin film transistors, or light emitting diodes. PMID:27219911

  6. Ultrastructural analysis of aminoglycoside-induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response.

    OpenAIRE

    Owens, Kelly,; Cunningham, Dale,; Macdonald, Glen; Rubel, Edwin,; Raible, David,; Pujol, Remy

    2007-01-01

    Loss of the mechanosensory hair cells in the auditory and vestibular organs leads to hearing and balance deficits. To investigate initial, in vivo events in aminoglycoside-induced hair cell damage, we examined hair cells from the lateral line of the zebrafish, Danio rerio. The mechanosensory lateral line is located externally on the animal and therefore allows direct manipulation and observation of hair cells. Labeling with vital dyes revealed a rapid response of hair cells to the aminoglycos...

  7. An integrative genomic and transcriptomic analysis reveals potential targets associated with cell proliferation in uterine leiomyomas

    DEFF Research Database (Denmark)

    Cirilo, Priscila Daniele Ramos; Marchi, Fábio Albuquerque; Barros Filho, Mateus de Camargo;

    2013-01-01

    integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell...... transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs....

  8. Genetic interaction maps in Escherichia coli reveal functional crosstalk among cell envelope biogenesis pathways.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    2011-11-01

    Full Text Available As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium and prototrophic (minimal medium culture conditions. The differential patterns of genetic interactions detected among > 235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens and an important target.

  9. Metagenomics, metatranscriptomics and single cell genomics reveal functional response of active Oceanospirillales to Gulf oil spill

    Energy Technology Data Exchange (ETDEWEB)

    Mason, Olivia U.; Hazen, Terry C.; Borglin, Sharon; Chain, Patrick S. G.; Dubinsky, Eric A.; Fortney, Julian L.; Han, James; Holman, Hoi-Ying N.; Hultman, Jenni; Lamendella, Regina; Mackelprang, Rachel; Malfatti, Stephanie; Tom, Lauren M.; Tringe, Susannah G.; Woyke, Tanja; Zhou, Jizhong; Rubin, Edward M.; Jansson, Janet K.

    2012-06-12

    The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

  10. Retrieval of the vacuolar H-ATPase from phagosomes revealed by live cell imaging.

    Directory of Open Access Journals (Sweden)

    Margaret Clarke

    Full Text Available BACKGROUND: The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized. METHODOLOGY: To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins. PRINCIPAL FINDINGS: We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved. CONCLUSIONS/SIGNIFICANCE: Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.

  11. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.

    Science.gov (United States)

    Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas; Lyon, David; Mullari, Meeli; Madsen, Maria V; Daniel, Jeremy A; Jensen, Lars J; Nielsen, Michael L

    2016-01-01

    The posttranslational modification of proteins by arginine methylation is functionally important, yet the breadth of this modification is not well characterized. Using high-resolution mass spectrometry, we identified 8030 arginine methylation sites within 3300 human proteins in human embryonic kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified by methylation. Through quantitative proteomics and RNA interference to examine arginine methylation stoichiometry, we unexpectedly found that the protein arginine methyltransferase (PRMT) family of arginine methyltransferases catalyzed methylation independently of arginine sequence context. In contrast to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially regulated the functions of the pre-mRNA splicing factor SRSF2 (serine/arginine-rich splicing factor 2) and the RNA transport ribonucleoprotein HNRNPUL1 (heterogeneous nuclear ribonucleoprotein U-like 1). Knocking down PRMT5 impaired the RNA binding function of SRSF2, whereas knocking down PRMT4 [also known as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human arginine methylome provides a missing piece in the global and integrative view of cellular physiology and protein regulation. PMID:27577262

  12. Visual cells remember earlier applied target: plasticity of orientation selectivity.

    Directory of Open Access Journals (Sweden)

    Narcis Ghisovan

    Full Text Available BACKGROUND: A canonical proposition states that, in mature brain, neurons responsive to sensory stimuli are tuned to specific properties installed shortly after birth. It is amply demonstrated that that neurons in adult visual cortex of cats are orientation-selective that is they respond with the highest firing rates to preferred oriented stimuli. METHODOLOGY/PRINCIPAL FINDINGS: In anesthetized cats, prepared in a conventional fashion for single cell recordings, the present investigation shows that presenting a stimulus uninterruptedly at a non-preferred orientation for twelve minutes induces changes in orientation preference. Across all conditions orientation tuning curves were investigated using a trial by trial method. Contrary to what has been previously reported with shorter adaptation duration, twelve minutes of adaptation induces mostly attractive shifts, i.e. toward the adapter. After a recovery period allowing neurons to restore their original orientation tuning curves, we carried out a second adaptation which produced three major results: (1 more frequent attractive shifts, (2 an increase of their magnitude, and (3 an additional enhancement of responses at the new or acquired preferred orientation. Additionally, we also show that the direction of shifts depends on the duration of the adaptation: shorter adaptation in most cases produces repulsive shifts, whereas adaptation exceeding nine minutes results in attractive shifts, in the same unit. Consequently, shifts in preferred orientation depend on the duration of adaptation. CONCLUSION/SIGNIFICANCE: The supplementary response improvements indicate that neurons in area 17 keep a memory trace of the previous stimulus properties, thereby upgrading cellular performance. It also highlights the dynamic nature of basic neuronal properties in adult cortex since repeated adaptations modified both the orientation tuning selectivity and the response strength to the preferred orientation. These

  13. Angiogenesis interactome and time course microarray data reveal the distinct activation patterns in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Liang-Hui Chu

    Full Text Available Angiogenesis involves stimulation of endothelial cells (EC by various cytokines and growth factors, but the signaling mechanisms are not completely understood. Combining dynamic gene expression time-course data for stimulated EC with protein-protein interactions associated with angiogenesis (the "angiome" could reveal how different stimuli result in different patterns of network activation and could implicate signaling intermediates as points for control or intervention. We constructed the protein-protein interaction networks of positive and negative regulation of angiogenesis comprising 367 and 245 proteins, respectively. We used five published gene expression datasets derived from in vitro assays using different types of blood endothelial cells stimulated by VEGFA (vascular endothelial growth factor A. We used the Short Time-series Expression Miner (STEM to identify significant temporal gene expression profiles. The statistically significant patterns between 2D fibronectin and 3D type I collagen substrates for telomerase-immortalized EC (TIME show that different substrates could influence the temporal gene activation patterns in the same cell line. We investigated the different activation patterns among 18 transmembrane tyrosine kinase receptors, and experimentally measured the protein level of the tyrosine-kinase receptors VEGFR1, VEGFR2 and VEGFR3 in human umbilical vein EC (HUVEC and human microvascular EC (MEC. The results show that VEGFR1-VEGFR2 levels are more closely coupled than VEGFR1-VEGFR3 or VEGFR2-VEGFR3 in HUVEC and MEC. This computational methodology can be extended to investigate other molecules or biological processes such as cell cycle.

  14. Single-cell analysis reveals a novel uncultivated magnetotactic bacterium within the candidate division OP3.

    Science.gov (United States)

    Kolinko, Sebastian; Jogler, Christian; Katzmann, Emanuel; Wanner, Gerhard; Peplies, Jörg; Schüler, Dirk

    2012-07-01

    Magnetotactic bacteria (MTB) are a diverse group of prokaryotes that orient along magnetic fields using membrane-coated magnetic nanocrystals of magnetite (Fe(3) O(4) ) or greigite (Fe(3) S(4) ), the magnetosomes. Previous phylogenetic analysis of MTB has been limited to few cultivated species and most abundant members of natural populations, which were assigned to Proteobacteria and the Nitrospirae phyla. Here, we describe a single cell-based approach that allowed the targeted phylogenetic and ultrastructural analysis of the magnetotactic bacterium SKK-01, which was low abundant in sediments of Lake Chiemsee. Morphologically conspicuous single cells of SKK-01 were micromanipulated from magnetically collected multi-species MTB populations, which was followed by whole genome amplification and ultrastructural analysis of sorted cells. Besides intracellular sulphur inclusions, the large ovoid cells of SKK-01 harbour ∼175 bullet-shaped magnetosomes arranged in multiple chains that consist of magnetite as revealed by TEM and EDX analysis. Sequence analysis of 16 and 23S rRNA genes from amplified genomic DNA as well as fluorescence in situ hybridization assigned SKK-01 to the candidate division OP3, which so far lacks any cultivated representatives. SKK-01 represents the first morphotype that can be assigned to the OP3 group as well as the first magnetotactic member of the PVC superphylum. PMID:22003954

  15. Combined in silico and in vivo analyses reveal role of Hes1 in taste cell differentiation.

    Directory of Open Access Journals (Sweden)

    Masato S Ota

    2009-04-01

    Full Text Available The sense of taste is of critical importance to animal survival. Although studies of taste signal transduction mechanisms have provided detailed information regarding taste receptor calcium signaling molecules (TRCSMs, required for sweet/bitter/umami taste signal transduction, the ontogeny of taste cells is still largely unknown. We used a novel approach to investigate the molecular regulation of taste system development in mice by combining in silico and in vivo analyses. After discovering that TRCSMs colocalized within developing circumvallate papillae (CVP, we used computational analysis of the upstream regulatory regions of TRCSMs to investigate the possibility of a common regulatory network for TRCSM transcription. Based on this analysis, we identified Hes1 as a likely common regulatory factor, and examined its function in vivo. Expression profile analyses revealed that decreased expression of nuclear HES1 correlated with expression of type II taste cell markers. After stage E18, the CVP of Hes1(-/ (- mutants displayed over 5-fold more TRCSM-immunoreactive cells than did the CVP of their wild-type littermates. Thus, according to our composite analyses, Hes1 is likely to play a role in orchestrating taste cell differentiation in developing taste buds.

  16. CAFET algorithm reveals Wnt/PCP signature in lung squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Yue Hu

    Full Text Available We analyzed the gene expression patterns of 138 Non-Small Cell Lung Cancer (NSCLC samples and developed a new algorithm called Coverage Analysis with Fisher's Exact Test (CAFET to identify molecular pathways that are differentially activated in squamous cell carcinoma (SCC and adenocarcinoma (AC subtypes. Analysis of the lung cancer samples demonstrated hierarchical clustering according to the histological subtype and revealed a strong enrichment for the Wnt signaling pathway components in the cluster consisting predominantly of SCC samples. The specific gene expression pattern observed correlated with enhanced activation of the Wnt Planar Cell Polarity (PCP pathway and inhibition of the canonical Wnt signaling branch. Further real time RT-PCR follow-up with additional primary tumor samples and lung cancer cell lines confirmed enrichment of Wnt/PCP pathway associated genes in the SCC subtype. Dysregulation of the canonical Wnt pathway, characterized by increased levels of β-catenin and epigenetic silencing of negative regulators, has been reported in adenocarcinoma of the lung. Our results suggest that SCC and AC utilize different branches of the Wnt pathway during oncogenesis.

  17. 21 CFR 866.6020 - Immunomagnetic circulating cancer cell selection and enumeration system.

    Science.gov (United States)

    2010-04-01

    ... cancer cells in a prepared sample of whole blood. This device is intended for adjunctive use in... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunomagnetic circulating cancer cell selection... Associated Antigen immunological Test Systems § 866.6020 Immunomagnetic circulating cancer cell selection...

  18. Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry.

    Science.gov (United States)

    Silva-Ayala, Daniela; López, Tomás; Gutiérrez, Michelle; Perrimon, Norbert; López, Susana; Arias, Carlos F

    2013-06-18

    Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the endosomal sorting complex required for transport (ESCRT) machinery were found. Here we provide evidence for a role of the ESCRT complex in the entry of simian and human RV strains in both monkey and human epithelial cells. In addition, the ESCRT-associated ATPase VPS4A and phospholipid lysobisphosphatidic acid, both crucial for the formation of intralumenal vesicles in multivesicular bodies, were also found to be required for cell entry. Interestingly, it seems that regardless of the molecules that rhesus RV and human RV strains use for cell-surface attachment and the distinct endocytic pathway used, all these viruses converge in early endosomes and use multivesicular bodies for cell entry. Furthermore, the small GTPases RHOA and CDC42, which regulate different types of clathrin-independent endocytosis, as well as early endosomal antigen 1 (EEA1), were found to be involved in this process. This work reports the direct involvement of the ESCRT machinery in the life cycle of a nonenveloped virus and highlights the complex mechanism that these viruses use to enter cells. It also illustrates the efficiency of high-throughput RNAi screenings as genetic tools for comprehensively studying the interaction between viruses and their host cells. PMID:23733942

  19. DNA-based digital tension probes reveal integrin forces during early cell adhesion

    Science.gov (United States)

    Zhang, Yun; Ge, Chenghao; Zhu, Cheng; Salaita, Khalid

    2014-10-01

    Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure because of a lack of methods for molecular force imaging. Here to address this need, we develop a new class of molecular tension probes that function as a switch to generate a 20- to 30-fold increase in fluorescence upon experiencing a threshold piconewton force. The probes employ immobilized DNA hairpins with tunable force response thresholds, ligands and fluorescence reporters. Quantitative imaging reveals that integrin tension is highly dynamic and increases with an increasing integrin density during adhesion formation. Mixtures of fluorophore-encoded probes show integrin mechanical preference for cyclized RGD over linear RGD peptides. Multiplexed probes with variable guanine-cytosine content within their hairpins reveal integrin preference for the more stable probes at the leading tip of growing adhesions near the cell edge. DNA-based tension probes are among the most sensitive optical force reporters to date, overcoming the force and spatial resolution limitations of traction force microscopy.

  20. Novel Cell Selection Proceduref LTE Hetnets Based on Mathematical Modelling of Proportional Fair Scheduling

    Directory of Open Access Journals (Sweden)

    Mohamed A. AboulHassan

    2013-12-01

    Full Text Available Femtocells have been considered one of the most imp ortant technologies in LTE networks to solve indoor coverage problem, however the randomness deployment of femtocells, leads to great challenge for select ing optimum serving cell. In this work, a new cell sele ction algorithm is proposed that enables new user t o select best serving cell whereas several factors ar e put into consideration other than highest instant aneous SNR or maximum RSRP such as cell load .A new predic tion algorithm is designed to predict the performance of (PF scheduling algorithm to calcula te expected number of RBs to be scheduled to new user, then reduction in achievable data rate due to both received SNR and instant cell load is estimat ed. The numerical results show that the new proposed ce ll selection algorithm achieves higher average cell throughput than conventional cell selection methods and achieves less cell load variance between diffe rent adjacent cells.

  1. CDK1 structures reveal conserved and unique features of the essential cell cycle CDK

    Science.gov (United States)

    Brown, Nicholas R.; Korolchuk, Svitlana; Martin, Mathew P.; Stanley, Will A.; Moukhametzianov, Rouslan; Noble, Martin E. M.; Endicott, Jane A.

    2015-04-01

    CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. Here we report the crystal structures of complexes of CDK1-Cks1 and CDK1-cyclin B-Cks2. These structures confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodelled by cyclin binding. Relative to CDK2-cyclin A, CDK1-cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation and shows differences in the substrate sequence features that determine activity. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We also describe the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors.

  2. Selective estrogen receptor modulators in T cell development and T cell dependent inflammation.

    Science.gov (United States)

    Bernardi, Angelina I; Andersson, Annica; Stubelius, Alexandra; Grahnemo, Louise; Carlsten, Hans; Islander, Ulrika

    2015-10-01

    Lasofoxifene (las) and bazedoxifene (bza) are third generation selective estrogen receptor modulators (SERMs) with minimal estrogenic side effects, approved for treatment of postmenopausal osteoporosis. T cells are involved in the pathology of postmenopausal osteoporosis and previous studies have established an important role for 17β-estradiol (E2) in T cell development and function. E2 causes a drastic thymic atrophy, alters the composition of thymic T cell populations, and inhibits T cell dependent inflammation. In contrast, the second generation SERM raloxifene (ral) lacks these properties. Although las and bza are drugs approved for treatment of postmenopausal bone loss, it is of importance to study their effects on other biological aspects in order to extend the potential use of these compounds. Therefore, the aim of this study was to investigate if treatment with las and bza affects T lymphopoiesis and T cell dependent inflammation. C57Bl6 mice were ovariectomized (ovx) and treated with vehicle, E2, ral, las or bza. As expected, E2 reduced both thymus weight and decreased the proportion of early T cell progenitors while increasing more mature T cell populations in the thymus. E2 also suppressed the T cell dependent delayed-type hypersensitivity (DTH) reaction to oxazolone (OXA). Ral and las, but not bza, decreased thymus weight, while none of the SERMs had any effects on T cell populations in the thymus or on inflammation in DTH. In conclusion, this study shows that treatment with las or bza does not affect T lymphopoiesis or T cell dependent inflammation. PMID:26044996

  3. Toward Cell Selective Surfaces: Cell Adhesion and Proliferation on Breath Figures with Antifouling Surface Chemistry.

    Science.gov (United States)

    Martínez-Campos, Enrique; Elzein, Tamara; Bejjani, Alice; García-Granda, Maria Jesús; Santos-Coquillat, Ana; Ramos, Viviana; Muñoz-Bonilla, Alexandra; Rodríguez-Hernández, Juan

    2016-03-16

    We report the preparation of microporous functional polymer surfaces that have been proven to be selective surfaces toward eukaryotic cells while maintaining antifouling properties against bacteria. The fabrication of functional porous films has been carried out by the breath figures approach that allowed us to create porous interfaces with either poly(ethylene glycol) methyl ether methacrylate (PEGMA) or 2,3,4,5,6-pentafluorostyrene (5FS). For this purpose, blends of block copolymers in a polystyrene homopolymer matrix have been employed. In contrast to the case of single functional polymer, using blends enables us to vary the chemical distribution of the functional groups inside and outside the formed pores. In particular, fluorinated groups were positioned at the edges while the hydrophilic PEGMA groups were selectively located inside the pores, as demonstrated by TOF-SIMS. More interestingly, studies of cell adhesion, growth, and proliferation on these surfaces confirmed that PEGMA functionalized interfaces are excellent candidates to selectively allow cell growth and proliferation while maintaining antifouling properties. PMID:26909529

  4. Functional malignant cell heterogeneity in pancreatic neuroendocrine tumors revealed by targeting of PDGF-DD

    Science.gov (United States)

    Cortez, Eliane; Gladh, Hanna; Braun, Sebastian; Bocci, Matteo; Cordero, Eugenia; Björkström, Niklas K.; Miyazaki, Hideki; Michael, Iacovos P.; Eriksson, Ulf; Folestad, Erika; Pietras, Kristian

    2016-01-01

    Intratumoral heterogeneity is an inherent feature of most human cancers and has profound implications for cancer therapy. As a result, there is an emergent need to explore previously unmapped mechanisms regulating distinct subpopulations of tumor cells and to understand their contribution to tumor progression and treatment response. Aberrant platelet-derived growth factor receptor beta (PDGFRβ) signaling in cancer has motivated the development of several antagonists currently in clinical use, including imatinib, sunitinib, and sorafenib. The discovery of a novel ligand for PDGFRβ, platelet-derived growth factor (PDGF)-DD, opened the possibility of a previously unidentified signaling pathway involved in tumor development. However, the precise function of PDGF-DD in tumor growth and invasion remains elusive. Here, making use of a newly generated Pdgfd knockout mouse, we reveal a functionally important malignant cell heterogeneity modulated by PDGF-DD signaling in pancreatic neuroendocrine tumors (PanNET). Our analyses demonstrate that tumor growth was delayed in the absence of signaling by PDGF-DD. Surprisingly, ablation of PDGF-DD did not affect the vasculature or stroma of PanNET; instead, we found that PDGF-DD stimulated bulk tumor cell proliferation by induction of paracrine mitogenic signaling between heterogeneous malignant cell clones, some of which expressed PDGFRβ. The presence of a subclonal population of tumor cells characterized by PDGFRβ expression was further validated in a cohort of human PanNET. In conclusion, we demonstrate a previously unrecognized heterogeneity in PanNET characterized by signaling through the PDGF-DD/PDGFRβ axis. PMID:26831065

  5. Solvatochromic Nile Red probes with FRET quencher reveal lipid order heterogeneity in living and apoptotic cells.

    Science.gov (United States)

    Kreder, Rémy; Pyrshev, Kyrylo A; Darwich, Zeinab; Kucherak, Oleksandr A; Mély, Yves; Klymchenko, Andrey S

    2015-06-19

    Detecting and imaging lipid microdomains (rafts) in cell membranes remain a challenge despite intensive research in the field. Two types of fluorescent probes are used for this purpose: one specifically labels a given phase (liquid ordered, Lo, or liquid disordered, Ld), while the other, being environment-sensitive (solvatochromic), stains the two phases in different emission colors. Here, we combined the two approaches by designing a phase-sensitive probe of the Ld phase and a quencher of the Ld phase. The former is an analogue of the recently developed Nile Red-based probe NR12S, bearing a bulky hydrophobic chain (bNR10S), while the latter is based on Black Hole Quencher-2 designed as bNR10S (bQ10S). Fluorescence spectroscopy of large unilamellar vesicles and microscopy of giant vesicles showed that the bNR10S probe can partition specifically into the Ld phase, while bQ10S can specifically quench the NR12S probe in the Ld phase so that only its fraction in the Lo phase remains fluorescent. Thus, the toolkit of two probes with quencher can specifically target Ld and Lo phases and identify their lipid order from the emission color. Application of this toolkit in living cells (HeLa, CHO, and 293T cell lines) revealed heterogeneity in the cell plasma membranes, observed as distinct probe environments close to the Lo and Ld phases of model membranes. In HeLa cells undergoing apoptosis, our toolkit showed the formation of separate domains of the Ld-like phase in the form of blebs. The developed tools open new possibilities in lipid raft research. PMID:25710589

  6. Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family

    International Nuclear Information System (INIS)

    Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the ∼1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family

  7. Stratification of alpha ganglion cells and ON/OFF directionally selective ganglion cells in the rabbit retina

    OpenAIRE

    Zhang, Jian; Li, Wei; HOSHI, HIDEO; Mills, Stephen L.; MASSEY, STEPHEN C.

    2005-01-01

    The correlation between cholinergic sensitivity and the level of stratification for ganglion cells was examined in the rabbit retina. As examples, we have used ON or OFF α ganglion cells and ON/OFF directionally selective (DS) ganglion cells. Nicotine, a cholinergic agonist, depolarized ON/OFF DS ganglion cells and greatly enhanced their firing rates but it had modest excitatory effects on ON or OFF α ganglion cells. As previously reported, we conclude that DS ganglion cells are the most sens...

  8. Bypassing antibiotic selection: positive screening of genetically modified cells with an antigen-dependent proliferation switch

    OpenAIRE

    Kawahara, Masahiro; Ueda, Hiroshi; Morita, Sumiyo; Tsumoto, Kouhei; Kumagai, Izumi; Nagamune, Teruyuki

    2003-01-01

    While antibiotic selection has been routinely used for the selection of genetically modified cells, administration of cytotoxic drugs often leads to deleterious effects not only to inert cells but also to transfected or transduced ones. In this study, we propose an Antigen-MEdiated Genetically modified cell Amplification (AMEGA) system employing antibody/receptor chimeras without antibiotic selection. Based on a rational design where the extracellular domains of dimeric erythropoietin recepto...

  9. Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells.

    Science.gov (United States)

    Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris; Kiessling, Ann A

    2016-01-15

    Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that

  10. Dissociation of internal energy-selected methyl bromide ion revealed from threshold photoelectron-photoion coincidence velocity imaging

    International Nuclear Information System (INIS)

    Dissociative photoionization of methyl bromide (CH3Br) in an excitation energy range of 10.45–16.90 eV has been investigated by using threshold photoelectron-photoion coincidence (TPEPICO) velocity imaging. The coincident time-of-flight mass spectra indicate that the ground state X2E of CH3Br+ is stable, and both A2A1 and B2E ionic excited states are fully dissociative to produce the unique fragment ion of CH3+. From TPEPICO 3D time-sliced velocity images of CH3+ dissociated from specific state-selected CH3Br+ ion, kinetic energy release distribution (KERD) and angular distribution of CH3+ fragment ion are directly obtained. Both spin-orbit states of Br(2P) atom can be clearly observed in fast dissociation of CH3Br+(A2A1) ion along C–Br rupture, while a KERD of Maxwell-Boltzmann profile is obtained in dissociation of CH3Br+(B2E) ion. With the aid of the re-calculated potential energy curves of CH3Br+ including spin-orbit coupling, dissociation mechanisms of CH3Br+ ion in A2A1 and B2E states along C–Br rupture are revealed. For CH3Br+(A2A1) ion, the CH3+ + Br(2P1/2) channel is occurred via an adiabatic dissociation by vibration, while the Br(2P3/2) formation is through vibronic coupling to the high vibrational level of X2E state followed by rapid dissociation. C–Br bond breaking of CH3Br+(B2E) ion can occur via slow internal conversion to the excited vibrational level of the lower electronic states and then dissociation

  11. Maitotoxin-induced cell death cascade in bovine aortic endothelial cells: divalent cation specificity and selectivity.

    Science.gov (United States)

    Wisnoskey, Brian J; Estacion, Mark; Schilling, William P

    2004-08-01

    The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs), a model for Ca(2+) overload-induced toxicity, reflects three sequential changes in plasmalemmal permeability. MTX initially activates Ca(2+)-permeable, nonselective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)). This is followed by the opening of large endogenous cytolytic/oncotic pores (COP) that allow molecules ionomycin and were significantly delayed in BAPTA-loaded cells. Experiments at the single-cell level revealed that Ba(2+) not only delayed the time to cell lysis but also caused desynchronization of the lytic phase. Last, membrane blebs, which were numerous and spherical in Ca(2+)-containing solutions, were poorly defined and greatly reduced in number in the presence of Ba(2+). Taken together, these results suggest that intracellular high-affinity Ca(2+)-binding proteins are involved in the MTX-induced changes in plasmalemmal permeability that are responsible for cell demise. PMID:15044153

  12. Live-cell microscopy reveals small molecule inhibitor effects on MAPK pathway dynamics.

    Directory of Open Access Journals (Sweden)

    Daniel J Anderson

    Full Text Available Oncogenic mutations in the mitogen activated protein kinase (MAPK pathway are prevalent in human tumors, making this pathway a target of drug development efforts. Recently, ATP-competitive Raf inhibitors were shown to cause MAPK pathway activation via Raf kinase priming in wild-type BRaf cells and tumors, highlighting the need for a thorough understanding of signaling in the context of small molecule kinase inhibitors. Here, we present critical improvements in cell-line engineering and image analysis coupled with automated image acquisition that allow for the simultaneous identification of cellular localization of multiple MAPK pathway components (KRas, CRaf, Mek1 and Erk2. We use these assays in a systematic study of the effect of small molecule inhibitors across the MAPK cascade either as single agents or in combination. Both Raf inhibitor priming as well as the release from negative feedback induced by Mek and Erk inhibitors cause translocation of CRaf to the plasma membrane via mechanisms that are additive in pathway activation. Analysis of Erk activation and sub-cellular localization upon inhibitor treatments reveals differential inhibition and activation with the Raf inhibitors AZD628 and GDC0879 respectively. Since both single agent and combination studies of Raf and Mek inhibitors are currently in the clinic, our assays provide valuable insight into their effects on MAPK signaling in live cells.

  13. Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

    Science.gov (United States)

    Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

    2015-10-13

    Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

  14. Quantitative phosphoproteomics reveals SLP-76 dependent regulation of PAG and Src family kinases in T cells.

    Directory of Open Access Journals (Sweden)

    Lulu Cao

    Full Text Available The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76 plays a critical scaffolding role in T cell receptor (TCR signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway.

  15. Dissecting the fission yeast regulatory network reveals phase-specific control elements of its cell cycle

    Directory of Open Access Journals (Sweden)

    Liu Liwen

    2009-09-01

    Full Text Available Abstract Background Fission yeast Schizosaccharomyces pombe and budding yeast Saccharomyces cerevisiae are among the original model organisms in the study of the cell-division cycle. Unlike budding yeast, no large-scale regulatory network has been constructed for fission yeast. It has only been partially characterized. As a result, important regulatory cascades in budding yeast have no known or complete counterpart in fission yeast. Results By integrating genome-wide data from multiple time course cell cycle microarray experiments we reconstructed a gene regulatory network. Based on the network, we discovered in addition to previously known regulatory hubs in M phase, a new putative regulatory hub in the form of the HMG box transcription factor SPBC19G7.04. Further, we inferred periodic activities of several less known transcription factors over the course of the cell cycle, identified over 500 putative regulatory targets and detected many new phase-specific and conserved cis-regulatory motifs. In particular, we show that SPBC19G7.04 has highly significant periodic activity that peaks in early M phase, which is coordinated with the late G2 activity of the forkhead transcription factor fkh2. Finally, using an enhanced Bayesian algorithm to co-cluster the expression data, we obtained 31 clusters of co-regulated genes 1 which constitute regulatory modules from different phases of the cell cycle, 2 whose phase order is coherent across the 10 time course experiments, and 3 which lead to identification of phase-specific control elements at both the transcriptional and post-transcriptional levels in S. pombe. In particular, the ribosome biogenesis clusters expressed in G2 phase reveal new, highly conserved RNA motifs. Conclusion Using a systems-level analysis of the phase-specific nature of the S. pombe cell cycle gene regulation, we have provided new testable evidence for post-transcriptional regulation in the G2 phase of the fission yeast cell cycle

  16. High overlap of CNVs and selection signatures revealed by varLD analyses of taurine and zebu cattle

    Science.gov (United States)

    Selection Signatures (SS) assessed through analysis of genomic data are being widely studied to discover population specific regions selected via artificial or natural selection. Different methodologies have been proposed for these analyses, each having specific limitations as to the age of the sele...

  17. Prolonged tamoxifen exposure selects a breast cancer cell clone that is stable in vitro and in vivo.

    Science.gov (United States)

    Sipila, P E; Wiebe, V J; Hubbard, G B; Koester, S K; Emshoff, V D; Maenpaa, J U; Wurz, G T; Seymour, R C; DeGregorio, M W

    1993-01-01

    The effects of long-term tamoxifen exposure on cell growth and cell cycle kinetics were compared between oestrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. In the MCF-7 cell line, prolonged tamoxifen exposure (0.5 mumol/l for > 100 days) blocked cells in G0-G1 of the cell cycle, and slowed the doubling time of cells from 30 to 59 h. These effects corresponded to an increase in the cellular accumulation of tamoxifen over time [mean area under concentration curve (AUC) = 77.92 mumoles/10(6)/cells/day]. In contrast, in the MDA-MB-231 cell line, long-term tamoxifen exposure had no obvious effect on the doubling time, and reduced cellular tamoxifen accumulation (mean AUC = 50.50 mumoles/10(6)/cells/day) compared to the MCF-7 cells. Flow cytometric analysis of MDA-MB-231 cells demonstrated that a new tetraploid clone emerged following 56 days of tamoxifen exposure. Inoculation of the MDA-MB-231 tetraploid clone and MDA-MB-231 wildtype cells into the opposite flanks of athymic nude mice resulted in the rapid growth of tetraploid tumours. The tetraploid tumours maintained their ploidy following tamoxifen treatment for nine consecutive serial transplantations. Histological examination of the fifth transplant generation xenografts revealed that the tetraploid tumour had a 25-30 times greater mass, area of haemorrhage and necrosis, a slightly higher mitotic index and was more anaplastic than the control neoplasm. The control wildtype MDA-MB-231 tumours maintained a stable ploidy following tamoxifen treatment until the eighth and ninth transplantation, when a tetraploid population appeared, suggesting that tamoxifen treatment may select for this clone in vivo. These studies suggest that prolonged tamoxifen exposure may select for new, stable, fast growing cell clones in vitro as well as in vivo. PMID:8297653

  18. Selective killing of cancer cells by nanoparticle-assisted ultrasound

    OpenAIRE

    Kosheleva, Olga K.; Lai, Tsung-Ching; Chen, Nelson G.; Hsiao, Michael; Chen, Chung-Hsuan

    2016-01-01

    Background Intense ultrasound, such as that used for tumor ablation, does not differentiate between cancerous and normal cells. A method combining ultrasound and biocompatible gold or magnetic nanoparticles (NPs) was developed under in vitro conditions using human breast and lung epithelial cells, which causes ultrasound to preferentially destroy cancerous cells. Results Co-cultures of BEAS-2B normal lung cells and A549 cancerous lung cells labeled with green and red fluorescent proteins, res...

  19. Quantitative trait loci mapping reveals candidate pathways regulating cell cycle duration in Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Siwo Geoffrey

    2010-10-01

    Full Text Available Abstract Background Elevated parasite biomass in the human red blood cells can lead to increased malaria morbidity. The genes and mechanisms regulating growth and development of Plasmodium falciparum through its erythrocytic cycle are not well understood. We previously showed that strains HB3 and Dd2 diverge in their proliferation rates, and here use quantitative trait loci mapping in 34 progeny from a cross between these parent clones along with integrative bioinformatics to identify genetic loci and candidate genes that control divergences in cell cycle duration. Results Genetic mapping of cell cycle duration revealed a four-locus genetic model, including a major genetic effect on chromosome 12, which accounts for 75% of the inherited phenotype variation. These QTL span 165 genes, the majority of which have no predicted function based on homology. We present a method to systematically prioritize candidate genes using the extensive sequence and transcriptional information available for the parent lines. Putative functions were assigned to the prioritized genes based on protein interaction networks and expression eQTL from our earlier study. DNA metabolism or antigenic variation functional categories were enriched among our prioritized candidate genes. Genes were then analyzed to determine if they interact with cyclins or other proteins known to be involved in the regulation of cell cycle. Conclusions We show that the divergent proliferation rate between a drug resistant and drug sensitive parent clone is under genetic regulation and is segregating as a complex trait in 34 progeny. We map a major locus along with additional secondary effects, and use the wealth of genome data to identify key candidate genes. Of particular interest are a nucleosome assembly protein (PFL0185c, a Zinc finger transcription factor (PFL0465c both on chromosome 12 and a ribosomal protein L7Ae-related on chromosome 4 (PFD0960c.

  20. Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yener Bülent

    2007-10-01

    Full Text Available Abstract Background Recently, we demonstrated that human mesenchymal stem cells (hMSC stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6: 1608–20, 2005. Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM proteins (collagen I, vitronectin, or laminin-5 or osteogenic media supplements (OS media. Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST, with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. Results Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. Conclusion The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

  1. Thymic selection of T-cell receptors as an extreme value problem

    CERN Document Server

    Kosmrlj, Andrej; Kardar, Mehran; Shakhnovich, Eugene I

    2009-01-01

    T lymphocytes (T cells) orchestrate adaptive immune responses upon activation. T cell activation requires sufficiently strong binding of T cell receptors (TCRs) on their surface to short peptides (p) derived from foreign proteins, which are bound to major histocompatibility (MHC) gene products (displayed on antigen presenting cells). A diverse and self-tolerant T cell repertoire is selected in the thymus. We map thymic selection processes to an extreme value problem and provide an analytic expression for the amino acid compositions of selected TCRs (which enable its recognition functions).

  2. Review of Adaptive Cell Selection Techniques in LTE-Advanced Heterogeneous Networks

    OpenAIRE

    Gadam, M. A.; Ahmed, Maryam Abdulazeez; Ng, Chee Kyun; Nordin, Nor Kamariah; Sali, Aduwati; Hashim, Fazirulhisyam

    2016-01-01

    Poor cell selection is the main challenge in Picocell (PeNB) deployment in Long Term Evolution- (LTE-) Advanced heterogeneous networks (HetNets) because it results in load imbalance and intercell interference. A selection technique based on cell range extension (CRE) has been proposed for LTE-Advanced HetNets to extend the coverage of PeNBs for load balancing. However, poor CRE bias setting in cell selection inhibits the attainment of desired cell splitting gains. By contrast, a cell selectio...

  3. Cell surface thiol isomerases may explain the platelet-selective action of S-nitrosoglutathione

    OpenAIRE

    Xiao, Fang; Gordge, Michael P

    2011-01-01

    S-nitrosoglutathione (GSNO) at low concentration inhibits platelet aggregation without causing vasodilation, suggesting platelet-selective nitric oxide delivery. The mechanism of this selectivity is unknown, but may involve cell surface thiol isomerases, in particular protein disulphide isomerase (csPDI) (EC 5.3.4.1). We have now compared csPDI expression and activity on platelets, endothelial cells and vascular smooth muscle cells, and the dependence on thiol reductase activity of these cell...

  4. Novel Cell Selection Proceduref LTE Hetnets Based on Mathematical Modelling of Proportional Fair Scheduling

    OpenAIRE

    Mohamed A. AboulHassan; Essam A. Sourour; Shawki Shaaban

    2013-01-01

    Femtocells have been considered one of the most imp ortant technologies in LTE networks to solve indoor coverage problem, however the randomness deployment of femtocells, leads to great challenge for select ing optimum serving cell. In this work, a new cell sele ction algorithm is proposed that enables new user t o select best serving cell whereas several factors ar e put into consideration other than highest instant aneous SNR or maximum RSRP such as cell loa...

  5. Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

    OpenAIRE

    Hirschberg, C B; Baker, R.M.; Perez, M.; Spencer, L A; Watson, D

    1981-01-01

    Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells ...

  6. Mechanosensory calcium-selective cation channels in epidermal cells

    Science.gov (United States)

    Ding, J. P.; Pickard, B. G.

    1993-01-01

    This paper explores the properties and likely functions of an epidermal Ca(2+)-selective cation channel complex activated by tension. As many as eight or nine linked or linkable equivalent conductance units or co-channels can open together. Open time for co-channel quadruplets and quintuplets tends to be relatively long with millimolar Mg2+ (but not millimolar Ca2+) at the cytosolic face of excised plasma membrane. Sensitivity to tension is regulated by transmembrane voltage and temperature. Under some circumstances channel activity is sychronized in rhythmic pulses. Certain lanthanides and a cytoskeleton-disturbing herbicide that inhibit gravitropic reception act on the channel system at low concentrations. Specifically, ethyl-N-phenylcarbamate promotes tension-dependent activity at micromolar levels. With moderate suction, Gd3+ provided at about 0.5 micromole at the extracellular face of the membrane promotes for several seconds but may then become inhibitory. Provision at 1-2 micromoles promotes and subsequently inhibits more vigorously (often abruptly and totally), and at high levels inhibits immediately. La3+, a poor gravitropic inhibitor, acts similarly but much more gradually and only at much higher concentrations. These properties, particularly these susceptibilities to modulation, indicate that in vivo the mechanosensitive channel must be mechanosensory and mechanoregulatory. It could serve to transduce the shear forces generated in the integrated wall-membrane-cytoskeleton system during turgor changes and cell expansion as well as transducing the stresses induced by gravity, touch and flexure. In so far as such transduction is modulated by voltage and temperature, the channels would also be sensors for these modalities as long as the wall-membrane-cytoskeleton system experiences mechanical stress.

  7. Crystal Structure of a Thermostable Alanine Racemase from Thermoanaerobacter tengcongensis MB4 Reveals the Role of Gln360 in Substrate Selection.

    Directory of Open Access Journals (Sweden)

    Xiaoliang Sun

    Full Text Available Pyridoxal 5'-phosphate (PLP dependent alanine racemase catalyzes racemization of L-Ala to D-Ala, a key component of the peptidoglycan network in bacterial cell wall. It has been extensively studied as an important antimicrobial drug target due to its restriction in eukaryotes. However, many marketed alanine racemase inhibitors also act on eukaryotic PLP-dependent enzymes and cause side effects. A thermostable alanine racemase (AlrTt from Thermoanaerobacter tengcongensis MB4 contains an evolutionarily non-conserved residue Gln360 in inner layer of the substrate entryway, which is supposed to be a key determinant in substrate specificity. Here we determined the crystal structure of AlrTt in complex with L-Ala at 2.7 Å resolution, and investigated the role of Gln360 by saturation mutagenesis and kinetic analysis. Compared to typical bacterial alanine racemase, presence of Gln360 and conformational changes of active site residues disrupted the hydrogen bonding interactions necessary for proper PLP immobilization, and decreased both the substrate affinity and turnover number of AlrTt. However, it could be complemented by introduction of hydrophobic amino acids at Gln360, through steric blocking and interactions with a hydrophobic patch near active site pocket. These observations explained the low racemase activity of AlrTt, revealed the essential role of Gln360 in substrate selection, and its preference for hydrophobic amino acids especially Tyr in bacterial alanine racemization. Our work will contribute new insights into the alanine racemization mechanism for antimicrobial drug development.

  8. Revealing nonergodic dynamics in living cells from a single particle trajectory

    Science.gov (United States)

    Lanoiselée, Yann; Grebenkov, Denis S.

    2016-05-01

    We propose the improved ergodicity and mixing estimators to identify nonergodic dynamics from a single particle trajectory. The estimators are based on the time-averaged characteristic function of the increments and can thus capture additional information on the process as compared to the conventional time-averaged mean-square displacement. The estimators are first investigated and validated for several models of anomalous diffusion, such as ergodic fractional Brownian motion and diffusion on percolating clusters, and nonergodic continuous-time random walks and scaled Brownian motion. The estimators are then applied to two sets of earlier published trajectories of mRNA molecules inside live Escherichia coli cells and of Kv2.1 potassium channels in the plasma membrane. These statistical tests did not reveal nonergodic features in the former set, while some trajectories of the latter set could be classified as nonergodic. Time averages along such trajectories are thus not representative and may be strongly misleading. Since the estimators do not rely on ensemble averages, the nonergodic features can be revealed separately for each trajectory, providing a more flexible and reliable analysis of single-particle tracking experiments in microbiology.

  9. Evolutionary strategies of cells and viruses in deep-sea hydrothermal systems revealed through comparative metagenomics

    Science.gov (United States)

    Anderson, R.; Sogin, M. L.; Baross, J. A.

    2013-12-01

    The deep-sea hydrothermal vent habitat hosts a diverse community of archaea and bacteria that withstand extreme fluctuations in environmental conditions. Abundant viruses in these systems must also withstand these environmental extremes, and a high proportion of viruses in these systems are lysogenic. Comparative analysis of a cellular and viral metagenome from a diffuse flow hydrothermal vent has provided insights into the evolutionary strategies of both cells and viruses in hydrothermal systems. We detected numerous mobile elements in the viral and cellular gene pools as well as a large number of prophage in the cellular fraction. We show that the hydrothermal vent viral gene pool is relatively enriched in genes related to energy metabolism, a feature that is unique to the hydrothermal vent viral gene pool compared to viral gene pools from other environments, indicating a potential for integrated prophage to enhance host metabolic flexibility. We also detected stronger purifying selection in the viral versus cellular gene pool, indicating selection pressures that promote prolonged viral integration in the host. Our results support the hypothesis that viruses enhance host genomic plasticity and adaptability in this extreme and dynamic environment. Finally, we will discuss general implications of this work for understanding the viral impact on biogeochemical cycles and evolutionary trajectories of microbial populations in the deep subsurface biosphere.

  10. Structures of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control

    Energy Technology Data Exchange (ETDEWEB)

    Burke, Jason R.; Hura, Greg L.; Rubin, Seth M. (UCSC); (LBNL)

    2012-07-18

    Cyclin-dependent kinase (Cdk) phosphorylation of the Retinoblastoma protein (Rb) drives cell proliferation through inhibition of Rb complexes with E2F transcription factors and other regulatory proteins. We present the first structures of phosphorylated Rb that reveal the mechanism of its inactivation. S608 phosphorylation orders a flexible 'pocket' domain loop such that it mimics and directly blocks E2F transactivation domain (E2F{sup TD}) binding. T373 phosphorylation induces a global conformational change that associates the pocket and N-terminal domains (RbN). This first multidomain Rb structure demonstrates a novel role for RbN in allosterically inhibiting the E2F{sup TD}-pocket association and protein binding to the pocket 'LxCxE' site. Together, these structures detail the regulatory mechanism for a canonical growth-repressive complex and provide a novel example of how multisite Cdk phosphorylation induces diverse structural changes to influence cell cycle signaling.

  11. Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation.

    Science.gov (United States)

    Chen, Xiaoying; Zhang, Kunshan; Zhou, Liqiang; Gao, Xinpei; Wang, Junbang; Yao, Yinan; He, Fei; Luo, Yuping; Yu, Yongchun; Li, Siguang; Cheng, Liming; Sun, Yi E

    2016-03-01

    The mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming various neural circuitries, through which sense, movement, thought, and emotion arise. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry properties possible. Here we report success in performing both electrophysiological and whole-genome transcriptome analyses on single human neurons in culture. Using Weighted Gene Coexpression Network Analyses (WGCNA), we identified gene clusters highly correlated with neuronal maturation judged by electrophysiological characteristics. A tight link between neuronal maturation and genes involved in ubiquitination and mitochondrial function was revealed. Moreover, we identified a list of candidate genes, which could potentially serve as biomarkers for neuronal maturation. Coupled electrophysiological recording and single cell transcriptome analysis will serve as powerful tools in the future to unveil molecular logics for neural circuitry functions. PMID:26883038

  12. Resource Competition Determines Selection of B Cell Repertoires

    OpenAIRE

    de Boer, R J; Freitas, A. A.; Perelson, A. S.

    2001-01-01

    Previous experiments with mouse chimeras demonstrated that cellular competition for antigen- specific survival signals plays a crucial role in the maintenance of the naive B cell repertoire. Transgenic (Tg) B cell populations in these chimeras have a shortened lifespan and poor competitive abilities as compared to more diverse non-Tg populations in the same mice. We develop a mathematical model to investigate the mechanism of B cell competition. The model allows for various B cell clones, gen...

  13. Selective internalization of self-assembled artificial oil bodies by HER2/neu-positive cells

    International Nuclear Information System (INIS)

    A novel delivery carrier was developed using artificial oil bodies (AOBs). Plant seed oil bodies (OBs) consist of a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with the storage protein oleosin (Ole). Ole consists of a central hydrophobic domain with two amphiphatic arms that extrude from the surface of OBs. In this study, a bivalent anti-HER2/neu affibody domain (ZH2) was fused with Ole at the C terminus. After overproduction in Escherichia coli, the fusion protein (Ole-ZH2) was recovered to assemble AOBs. The size of self-assembled AOBs was tailored by varying the oil/Ole-ZH2 ratio and pH to reach a nanoscale. Upon co-incubation with tumor cells, the nanoscale AOBs encapsulated with a hydrophobic fluorescence dye were selectively internalized by HER2/neu-overexpressing cells and displayed biocompatibility with the cells. In addition, the ZH2-mediated endosomal entry of AOBs occurred in a time- and AOB dose-dependent manner. The internalization efficiency was as high as 90%. The internalized AOBs disintegrated at the non-permissive pH (e.g. in acidic endosomes) and the cargo dye was released. Results of in vitro study revealed a sustained and prolonged release profile. Taken together, our findings indicate the potential of AOBs as a delivery carrier.

  14. Selective suppression of human papillomavirus transcription in non-tumorigenic cells by 5-azacytidine.

    Science.gov (United States)

    Rösl, F; Dürst, M; zur Hausen, H

    1988-01-01

    The transcription of human papillomavirus type 18 (HPV 18) is selectively suppressed in non-tumorigenic HeLa x fibroblast or HeLa x keratinocyte cell hybrids by 5-azacytidine. In contrast, viral gene expression is not influenced by 5-azacytidine in both tumorigenic hybrid segregants and in the parental HeLa cells. The suppression mechanism seems to operate at the level of initiation of transcription since nuclear run-on experiments show the absence of elongated nascent viral RNA, whereas the transcription of cellular reference genes remains unaffected. Down-regulation of HPV 18 mRNA correlates directly with cessation of cellular growth and can be abolished using the protein synthesis inhibitor cycloheximide. Furthermore human keratinocytes immortalized by HPV 16 but still retaining the non-tumorigenic phenotype reveal the same inhibitory effect on viral transcription after treatment with 5-azacytidine. These results support a model of a postulated intracellular control mechanism, directed against papillomavirus transcription, which can be induced by 5-azacytidine and appears to correlate with the presence of specific chromosomes in non-tumorigenic cells. Images PMID:2457495

  15. Development of Photoactivated Fluorescent N-Hydroxyoxindoles and Their Application for Cell-Selective Imaging.

    Science.gov (United States)

    Lai, Jinping; Yu, An; Yang, Letao; Zhang, Yixiao; Shah, Birju P; Lee, Ki Bum

    2016-04-25

    Photoactivatable fluorophores are essential tools for studying the dynamic molecular interactions within important biological systems with high spatiotemporal resolution. However, currently developed photoactivatable fluorophores based on conventional dyes have several limitations including reduced photoactivation efficiency, cytotoxicity, large molecular size, and complicated organic synthesis. To overcome these challenges, we herein report a class of photoactivatable fluorescent N-hydroxyoxindoles formed through the intramolecular photocyclization of substituted o-nitrophenyl ethanol (ONPE). These oxindole fluorophores afford excellent photoactivation efficiency with ultra-high fluorescence enhancement (up to 800-fold) and are small in size. Furthermore, the oxindole derivatives show exceptional biocompatibility by generating water as the only photolytic side product. Moreover, structure-activity relationship analysis clearly revealed the strong correlation between the fluorescent properties and the substituent groups, which can serve as a guideline for the further development of ONPE-based fluorescent probes with desired photophysical and biological properties. As a proof-of-concept, we demonstrated the capability of a new substituted ONPE that has an uncaging wavelength of 365-405 nm and an excitation/emission at 515 and 620 nm, for the selective imaging of a cancer cell line (Hela cells) and a human neural stem cell line (hNSCs). PMID:27004772

  16. Selective internalization of self-assembled artificial oil bodies by HER2/neu-positive cells

    Science.gov (United States)

    Chiang, Chung-Jen; Lin, Li-Jen; Lin, Che-Chin; Chang, Chih-Hsiang; Chao, Yun-Peng

    2011-01-01

    A novel delivery carrier was developed using artificial oil bodies (AOBs). Plant seed oil bodies (OBs) consist of a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with the storage protein oleosin (Ole). Ole consists of a central hydrophobic domain with two amphiphatic arms that extrude from the surface of OBs. In this study, a bivalent anti-HER2/neu affibody domain (ZH2) was fused with Ole at the C terminus. After overproduction in Escherichia coli, the fusion protein (Ole-ZH2) was recovered to assemble AOBs. The size of self-assembled AOBs was tailored by varying the oil/Ole-ZH2 ratio and pH to reach a nanoscale. Upon co-incubation with tumor cells, the nanoscale AOBs encapsulated with a hydrophobic fluorescence dye were selectively internalized by HER2/neu-overexpressing cells and displayed biocompatibility with the cells. In addition, the ZH2-mediated endosomal entry of AOBs occurred in a time- and AOB dose-dependent manner. The internalization efficiency was as high as 90%. The internalized AOBs disintegrated at the non-permissive pH (e.g. in acidic endosomes) and the cargo dye was released. Results of in vitro study revealed a sustained and prolonged release profile. Taken together, our findings indicate the potential of AOBs as a delivery carrier.

  17. Selective internalization of self-assembled artificial oil bodies by HER2/neu-positive cells

    Energy Technology Data Exchange (ETDEWEB)

    Chiang, Chung-Jen; Lin, Che-Chin [Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan (China); Lin, Li-Jen [School of Chinese Medicine, China Medical University, Taichung, Taiwan (China); Chang, Chih-Hsiang; Chao, Yun-Peng, E-mail: cjchiang@mail.cmu.edu.tw, E-mail: ypchao@fcu.edu.tw [Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan (China)

    2011-01-07

    A novel delivery carrier was developed using artificial oil bodies (AOBs). Plant seed oil bodies (OBs) consist of a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with the storage protein oleosin (Ole). Ole consists of a central hydrophobic domain with two amphiphatic arms that extrude from the surface of OBs. In this study, a bivalent anti-HER2/neu affibody domain (ZH2) was fused with Ole at the C terminus. After overproduction in Escherichia coli, the fusion protein (Ole-ZH2) was recovered to assemble AOBs. The size of self-assembled AOBs was tailored by varying the oil/Ole-ZH2 ratio and pH to reach a nanoscale. Upon co-incubation with tumor cells, the nanoscale AOBs encapsulated with a hydrophobic fluorescence dye were selectively internalized by HER2/neu-overexpressing cells and displayed biocompatibility with the cells. In addition, the ZH2-mediated endosomal entry of AOBs occurred in a time- and AOB dose-dependent manner. The internalization efficiency was as high as 90%. The internalized AOBs disintegrated at the non-permissive pH (e.g. in acidic endosomes) and the cargo dye was released. Results of in vitro study revealed a sustained and prolonged release profile. Taken together, our findings indicate the potential of AOBs as a delivery carrier.

  18. Gestational lead exposure selectively decreases retinal dopamine amacrine cells and dopamine content in adult mice

    International Nuclear Information System (INIS)

    Gestational lead exposure (GLE) produces supernormal scotopic electroretinograms (ERG) in children, monkeys and rats, and a novel retinal phenotype characterized by an increased number of rod photoreceptors and bipolar cells in adult mice and rats. Since the loss of dopaminergic amacrine cells (DA ACs) in GLE monkeys and rats contributes to supernormal ERGs, the retinal DA system was analyzed in mice following GLE. C57BL/6 female mice were exposed to low (27 ppm), moderate (55 ppm) or high (109 ppm) lead throughout gestation and until postnatal day 10 (PN10). Blood [Pb] in control, low-, moderate- and high-dose GLE was ≤ 1, ≤ 10, ∼ 25 and ∼ 40 μg/dL, respectively, on PN10 and by PN30 all were ≤ 1 μg/dL. At PN60, confocal-stereology studies used vertical sections and wholemounts to characterize tyrosine hydroxylase (TH) expression and the number of DA and other ACs. GLE dose-dependently and selectively decreased the number of TH-immunoreactive (IR) DA ACs and their synaptic plexus without affecting GABAergic, glycinergic or cholinergic ACs. Immunoblots and confocal revealed dose-dependent decreases in retinal TH protein expression and content, although monoamine oxidase-A protein and gene expression were unchanged. High-pressure liquid chromatography showed that GLE dose-dependently decreased retinal DA content, its metabolites and DA utilization/release. The mechanism of DA selective vulnerability is unknown. However, a GLE-induced loss/dysfunction of DA ACs during development could increase the number of rods and bipolar cells since DA helps regulate neuronal proliferation, whereas during adulthood it could produce ERG supernormality as well as altered circadian rhythms, dark/light adaptation and spatial contrast sensitivity. -- Highlights: ► Peak [BPb] in control, low-, moderate- and high-dose newborn mice with gestational lead exposure: ≤ 1, ≤ 10, 25 and 40 μg/dL ► Gestational lead exposure dose-dependently decreased the number of TH

  19. Gestational lead exposure selectively decreases retinal dopamine amacrine cells and dopamine content in adult mice

    Energy Technology Data Exchange (ETDEWEB)

    Fox, Donald A., E-mail: dafox@uh.edu [College of Optometry, University of Houston, Houston, TX (United States); Department of Biology and Biochemistry, University of Houston, Houston, TX (United States); Department of Pharmacology and Pharmaceutical Sciences, University of Houston, Houston, TX (United States); Hamilton, W. Ryan [Department of Biology and Biochemistry, University of Houston, Houston, TX (United States); Johnson, Jerry E. [Department of Natural Sciences, University of Houston-Downtown, Houston, TX (United States); Xiao, Weimin [College of Optometry, University of Houston, Houston, TX (United States); Chaney, Shawntay; Mukherjee, Shradha [Department of Biology and Biochemistry, University of Houston, Houston, TX (United States); Miller, Diane B.; O' Callaghan, James P. [Toxicology and Molecular Biology Branch, Health Effects Research Laboratory, Centers for Disease Control and Prevention-NIOSH, Morgantown, WV USA (United States)

    2011-11-15

    Gestational lead exposure (GLE) produces supernormal scotopic electroretinograms (ERG) in children, monkeys and rats, and a novel retinal phenotype characterized by an increased number of rod photoreceptors and bipolar cells in adult mice and rats. Since the loss of dopaminergic amacrine cells (DA ACs) in GLE monkeys and rats contributes to supernormal ERGs, the retinal DA system was analyzed in mice following GLE. C57BL/6 female mice were exposed to low (27 ppm), moderate (55 ppm) or high (109 ppm) lead throughout gestation and until postnatal day 10 (PN10). Blood [Pb] in control, low-, moderate- and high-dose GLE was {<=} 1, {<=} 10, {approx} 25 and {approx} 40 {mu}g/dL, respectively, on PN10 and by PN30 all were {<=} 1 {mu}g/dL. At PN60, confocal-stereology studies used vertical sections and wholemounts to characterize tyrosine hydroxylase (TH) expression and the number of DA and other ACs. GLE dose-dependently and selectively decreased the number of TH-immunoreactive (IR) DA ACs and their synaptic plexus without affecting GABAergic, glycinergic or cholinergic ACs. Immunoblots and confocal revealed dose-dependent decreases in retinal TH protein expression and content, although monoamine oxidase-A protein and gene expression were unchanged. High-pressure liquid chromatography showed that GLE dose-dependently decreased retinal DA content, its metabolites and DA utilization/release. The mechanism of DA selective vulnerability is unknown. However, a GLE-induced loss/dysfunction of DA ACs during development could increase the number of rods and bipolar cells since DA helps regulate neuronal proliferation, whereas during adulthood it could produce ERG supernormality as well as altered circadian rhythms, dark/light adaptation and spatial contrast sensitivity. -- Highlights: Black-Right-Pointing-Pointer Peak [BPb] in control, low-, moderate- and high-dose newborn mice with gestational lead exposure: {<=} 1, {<=} 10, 25 and 40 {mu}g/dL Black

  20. Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane.

    Science.gov (United States)

    Ferreira, Savio Siqueira; Hotta, Carlos Takeshi; Poelking, Viviane Guzzo de Carli; Leite, Debora Chaves Coelho; Buckeridge, Marcos Silveira; Loureiro, Marcelo Ehlers; Barbosa, Marcio Henrique Pereira; Carneiro, Monalisa Sampaio; Souza, Glaucia Mendes

    2016-05-01

    Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production. PMID:26820137

  1. Microselection - Affinity Selecting Antibodies against a Single Rare Cell in a Heterogeneous Population

    DEFF Research Database (Denmark)

    Sørensen, Morten Draeby; Agerholm, Inge Errebo; Christensen, Britta; Kølvraa, Steen; Kristensen, Peter

    2010-01-01

    antibodies. Here we have generated a microselection method allowing antibody selection, by phage display, targeting a single cell in a heterogeneous population. One K562 cell (female origin) was positioned on glass-slide among millions of lymphocytes from male donor, identifying the K562 cell by FISH (XX......). Several single cell selections were performed on such individual slides. The phage particles bound to the target cell is protected by a minute disc, while inactivating all remaining phage by UV-irradiation; leaving only the phage bound to the target cell viable. We hereby retrieved up to eight antibodies...

  2. Selective cell adhesion inhibitors: Barbituric acid based alpha4beta7--MAdCAM inhibitors.

    Science.gov (United States)

    Harriman, Geraldine C; Brewer, Matthias; Bennett, Robert; Kuhn, Cyrille; Bazin, Marc; Larosa, Greg; Skerker, Paul; Cochran, Nancy; Gallant, Debra; Baxter, Deborah; Picarella, Dominic; Jaffee, Bruce; Luly, Jay R; Briskin, Michael J

    2008-04-01

    A novel series of barbituric acid derivatives were identified as selective inhibitors of alpha4beta7 MAdCAM (mucosal addressin cell adhesion molecule-1) interactions via a high throughput screening exercise. These inhibitors were optimized to submicromolar potencies in whole cell adhesion assays, retaining their selectivity over alpha4beta1 VCAM. PMID:18331794

  3. DNA methylation analysis reveals distinct methylation signatures in pediatric germ cell tumors

    International Nuclear Information System (INIS)

    Aberrant DNA methylation is a prominent feature of many cancers, and may be especially relevant in germ cell tumors (GCTs) due to the extensive epigenetic reprogramming that occurs in the germ line during normal development. We used the Illumina GoldenGate Cancer Methylation Panel to compare DNA methylation in the three main histologic subtypes of pediatric GCTs (germinoma, teratoma and yolk sac tumor (YST); N = 51) and used recursively partitioned mixture models (RPMM) to test associations between methylation pattern and tumor and demographic characteristics. We identified genes and pathways that were differentially methylated using generalized linear models and Ingenuity Pathway Analysis. We also measured global DNA methylation at LINE1 elements and evaluated methylation at selected imprinted loci using pyrosequencing. Methylation patterns differed by tumor histology, with 18/19 YSTs forming a distinct methylation class. Four pathways showed significant enrichment for YSTs, including a human embryonic stem cell pluripotency pathway. We identified 190 CpG loci with significant methylation differences in mature and immature teratomas (q < 0.05), including a number of CpGs in stem cell and pluripotency-related pathways. Both YST and germinoma showed significantly lower methylation at LINE1 elements compared with normal adjacent tissue while there was no difference between teratoma (mature and immature) and normal tissue. DNA methylation at imprinted loci differed significantly by tumor histology and location. Understanding methylation patterns may identify the developmental stage at which the GCT arose and the at-risk period when environmental exposures could be most harmful. Further, identification of relevant genetic pathways could lead to the development of new targets for therapy

  4. Genome wide analysis of the world's sheep breeds reveals high levels of historic mixture and strong recent selection

    Science.gov (United States)

    Through their domestication and subsequent selection, sheep have been adapted to thrive in a diverse range of environments. To characterize the genetic consequence of both domestication and selection, we genotyped 49,034 single nucleotide polymorphisms in 2,819 animals from a diverse collection of ...

  5. Selective adhesion of intestinal epithelial cells on patterned films with amine functionalities formed by plasma enhanced chemical vapor deposition

    International Nuclear Information System (INIS)

    Control of cell adhesion to surfaces is important to develop analytical tools in the areas of biomedical engineering. To control cell adhesiveness of the surface, we constructed a variety of plasma polymerized hexamethyldisiloxane (PPHMDSO) thin films deposited at the plasma power range of 10-100 W by plasma enhanced chemical vapor deposition (PECVD). The PPHMDSO film that was formed at 10 W was revealed to be resistant to cell adhesion. The resistance to cell adhesion is closely related to physicochemical properties of the film. Atomic force microscopic data show an increase in surface roughness from 0.52 nm to 0.74 nm with increasing plasma power. From Fourier transform infrared (FT-IR) absorption spectroscopy data, it was also determined that the methyl (-CH3) peak intensity increases with increasing plasma power, whereas the hydroxyl (-OH) peak decreases. X-ray photoelectron spectroscopy data reveal an increase in C-O bonding with increasing plasma power. These results suggest that C-O bonding and hydroxyl (-OH) and methyl (-CH3) functional groups play a critical part in cell adhesion. Furthermore, to enhance a diversity of film surface, we accumulated the patterned plasma polymerized ethylenediamine (PPEDA) thin film on the top of the PPHMDSO thin film. The PPEDA film is established to be strongly cell-adherent. This patterned two-layer film stacking method can be used to form the selectively limited cell-adhesive PPEDA spots over the adhesion-resistant surface.

  6. New Strategies for Designing Inexpensive but Selective Bioadsorbants for Environmental Pollutants: Selection of specific Ligands and Their Cell Surface Expression

    Energy Technology Data Exchange (ETDEWEB)

    Brent L. Iverson; George Georgiou; Mohammad M. Ataai; Richard R. Koepsel

    2001-02-22

    The Broad, long term objective of the research plan is to develop exquisitely selective polypeptide metal chelators for the remediation of aqueous systems. A variety of polypeptide chelators will be developed and optimized ranging from antibodies to small peptides. Then, through unique molecular engineering approaches developed in our laboratories, the polypeptide chelators will be anchored directly on the surface of the cells that produce them. Thus, instead of using isolated biomolecules we will employ inexpensive genetically engineered whole cell adsorbents. Following a simple, easily scaleable treatment, the engineered cells can be used to manufacture an inexpensive, particulate adsorbent for metal removal.

  7. Resveratrol engages selective apoptotic signals in gastric adenocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    William L Riles; Jason Erickson; Sanjay Nayyar; Mary Jo Atten; Bashar M Attar; Oksana Holian

    2006-01-01

    AIM: To investigate the intracellular apoptotic signals engaged by resveratrol in three gastric adenocarcinoma cancer cell lines, two of which (AGS and SNU-1) express p53 and one (KATO-Ⅲ) with deleted p53.METHODS: Nuclear fragmentation was used to quantitate apoptotic cells; caspase activity was determined by photometric detection of cleaved substrates; formation of oxidized cytochrome C was used to measure cytochrome C activity, and Western blot analysis was used to determine protein expression.RESULTS: Gastric cancer cells, irrespective of their p53 status, responded to resveratrol with fragmentation of DNA and cleavage of nuclear lamins A and B and PARP, Resveratrol, however, has no effect on mitochondria-associated apoptotic proteins Bcl-2, Bclxl, Bax, Bid or Smac/Diablo, and did not promote subcellular redistribution of cytochrome C, indicating that resveratrol-induced apoptosis of gastric carcinoma cells does not require breakdown of mitochondrial membrane integrity. Resveratrol up-regulated p53 protein in SNU-1 and AGS cells but there was a difference in response of intracellular apoptotic signals between these cell lines.SNU-1 cells responded to resveratrol treatment with down-regulation of survivin, whereas in AGS and KATO-Ⅲ cells resveratrol stimulated caspase 3 and cytochrome C oxidase activities.CONCLUSION: These findings indicate that even within a specific cancer the intracellular apoptotic signals engaged by resveratrol are cell type dependent and suggest that such differences may be related to differentiation or lack of differentiation of these cells.

  8. Revealing changes in molecular composition of plant cell walls on the micron-level by Raman mapping and vertex component analysis (VCA

    Directory of Open Access Journals (Sweden)

    Notburga eGierlinger

    2014-06-01

    Full Text Available At the molecular level the plant cell walls consist of a few nanometer thick semi-crystalline cellulose fibrils embedded in amorphous matrix polymers such as pectins, hemicelluloses and lignins. The arrangement of these molecules within the cell wall in different plant tissues, cells and cell wall layers is of crucial importance for a better understanding and thus optimized utilization of plant biomass. During the last years Confocal Raman microscopy evolved as a powerful method in plant science by revealing the different molecules in context with the microstructure. In this study two-dimensional spectral maps have been acquired of micro-cross-sections of spruce (softwood and beech (hardwood. Raman images have been derived by using univariate (band integration, height ratios and multivariate methods (vertex component analysis, VCA. While univariate analysis only visualizes changes in selected band heights or areas, VCA separates anatomical regions and cell wall layers with the most different molecular structures by projecting the data to the identified orthogonal subspace in an interactive way and finding the endmember by repeated iteration. Beside visualization of the distinguished regions and features the underlying molecular structure can be derived based on the endmember spectra. Only one pure component spectrum (lignin from the cell corner was extracted, while all other endmember spectra represented mixtures characteristic for the different resolved spatial areas. VCA revealed that the lumen sided S3 layer has a similar molecular composition as the pit membrane, both revealing a clear change in lignin composition compared to all other cell wall regions. Within the S2 layer a lamellar structure was visualized, which was elucidated to derive also from slight changes in lignin composition and content and might be due to successive but not uniform lignification during growth.

  9. Selective assembly of laminin variants by human carcinoma cells

    DEFF Research Database (Denmark)

    Wewer, U M; Wayner, E A; Hoffstrom, B G;

    1994-01-01

    in negligible amounts as detected by Northern blotting and PCR. The only exception was the HU-1 lung adenocarcinoma cell line which expressed significant quantities of laminin M chain mRNA and lower levels of laminin A chain mRNA. The presence in the HU-1 cells of translated polypeptides was...... chain. CONCLUSIONS: All human carcinoma cell lines produced laminin chains B1, B2 and S, but no or little A or M. The only exception was the lung carcinoma cell line HU-1. Human HU-1 carcinoma cells in culture synthesize several homologous laminin chains and regulate the process of assembly, secretion......BACKGROUND: The laminins are heterotrimeric basement membrane glycoproteins. Eight subunits that can be assembled into laminins have been characterized and are known as: A, B1, B2, S, M, K, B2t, B1k laminin chains. Although many neoplastic cells secrete laminins and some of them even assemble...

  10. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, M.

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution imaging techniques. Also, translating findings between model substrates to intact biomass is critical for evaluating enzyme performance. Here we employ a fungal free enzyme cocktail, a complexed cellulosomal system, and a combination of the two to investigate saccharification mechanisms on cellulose I, II and III along with corn stover from Clean Fractionation (CF), which is an Organosolv pretreatment. The insoluble Cellulose Enriched Fraction (CEF) from CF contains mainly cellulose with minor amounts of residual hemicellulose and lignin, the amount of which depends on the CF pretreatment severity. Enzymatic digestions at both low and high-solids loadings demonstrate that CF reduces the amount of enzyme required to depolymerize polysaccharides relative to deacetylated, dilute acid pretreated corn stover. Transmission and scanning electron microscopy of the biomass provides evidence for the different mechanisms of enzymatic deconstruction between free and complexed enzyme systems, and reveals the basis for the synergistic relationship between the two enzyme paradigms on a process-relevant substrate for the first time. These results also demonstrate that the presence of lignin, rather than cellulose morphology, is more detrimental to cellulosome action than to free cellulases. As enzyme costs are a major economic driver for biorefineries, this study provides key inputs for the evaluation of CF as a pretreatment method for biomass conversion.

  11. Whole-Genome Sequencing Reveals Diverse Models of Structural Variations in Esophageal Squamous Cell Carcinoma.

    Science.gov (United States)

    Cheng, Caixia; Zhou, Yong; Li, Hongyi; Xiong, Teng; Li, Shuaicheng; Bi, Yanghui; Kong, Pengzhou; Wang, Fang; Cui, Heyang; Li, Yaoping; Fang, Xiaodong; Yan, Ting; Li, Yike; Wang, Juan; Yang, Bin; Zhang, Ling; Jia, Zhiwu; Song, Bin; Hu, Xiaoling; Yang, Jie; Qiu, Haile; Zhang, Gehong; Liu, Jing; Xu, Enwei; Shi, Ruyi; Zhang, Yanyan; Liu, Haiyan; He, Chanting; Zhao, Zhenxiang; Qian, Yu; Rong, Ruizhou; Han, Zhiwei; Zhang, Yanlin; Luo, Wen; Wang, Jiaqian; Peng, Shaoliang; Yang, Xukui; Li, Xiangchun; Li, Lin; Fang, Hu; Liu, Xingmin; Ma, Li; Chen, Yunqing; Guo, Shiping; Chen, Xing; Xi, Yanfeng; Li, Guodong; Liang, Jianfang; Yang, Xiaofeng; Guo, Jiansheng; Jia, JunMei; Li, Qingshan; Cheng, Xiaolong; Zhan, Qimin; Cui, Yongping

    2016-02-01

    Comprehensive identification of somatic structural variations (SVs) and understanding their mutational mechanisms in cancer might contribute to understanding biological differences and help to identify new therapeutic targets. Unfortunately, characterization of complex SVs across the whole genome and the mutational mechanisms underlying esophageal squamous cell carcinoma (ESCC) is largely unclear. To define a comprehensive catalog of somatic SVs, affected target genes, and their underlying mechanisms in ESCC, we re-analyzed whole-genome sequencing (WGS) data from 31 ESCCs using Meerkat algorithm to predict somatic SVs and Patchwork to determine copy-number changes. We found deletions and translocations with NHEJ and alt-EJ signature as the dominant SV types, and 16% of deletions were complex deletions. SVs frequently led to disruption of cancer-associated genes (e.g., CDKN2A and NOTCH1) with different mutational mechanisms. Moreover, chromothripsis, kataegis, and breakage-fusion-bridge (BFB) were identified as contributing to locally mis-arranged chromosomes that occurred in 55% of ESCCs. These genomic catastrophes led to amplification of oncogene through chromothripsis-derived double-minute chromosome formation (e.g., FGFR1 and LETM2) or BFB-affected chromosomes (e.g., CCND1, EGFR, ERBB2, MMPs, and MYC), with approximately 30% of ESCCs harboring BFB-derived CCND1 amplification. Furthermore, analyses of copy-number alterations reveal high frequency of whole-genome duplication (WGD) and recurrent focal amplification of CDCA7 that might act as a potential oncogene in ESCC. Our findings reveal molecular defects such as chromothripsis and BFB in malignant transformation of ESCCs and demonstrate diverse models of SVs-derived target genes in ESCCs. These genome-wide SV profiles and their underlying mechanisms provide preventive, diagnostic, and therapeutic implications for ESCCs. PMID:26833333

  12. Effect of selected insecticides on SF9 insect cell line

    International Nuclear Information System (INIS)

    The toxic effect of three insecticides: dimethoate (organophosphate insecticide), acetamiprid (neonicotinoid insecticide) and deltamethrin (pyrethroid insecticide) were evaluated in vitro on cultured Sf9 cell line. Cell growth inhibition was measured by the 3- (4,5- dimethylthiazol - 2-yl) - 2,5 - diphenyl tetrazolium bromide (MTT) assay. Regression Analysis was used to estimate the 20% inhibition of cells growth (IC 20). The IC 20 values obtained for deltamethrin, acetamipridand dimethoate were: 46.8, 61.6 and 68.9 μM, respectively. The proportion of phagocytic cells was positively correlated with the applied concentrations of the insecticides. (author)

  13. Selective cytotoxicity of benzyl isothiocyanate in the proliferating fibroblastoid cells.

    Science.gov (United States)

    Miyoshi, Noriyuki; Uchida, Koji; Osawa, Toshihiko; Nakamura, Yoshimasa

    2007-02-01

    In the present study, experiments using presynchronization culture cells demonstrated that benzyl ITC (BITC), previously isolated from a tropical papaya fruit extract, induced the cytotoxic effect preferentially in the proliferating human colon CCD-18Co cells to the quiescent ones. Quiescent CCD-18Co cells were virtually unaffected by BITC and marginal cytotoxicity was observed at 15 microM. We observed that BITC dramatically induced the p53 phosphorylation and stabilization only in the quiescent (G(0)/G(1) phase-arrested) cells, but not significantly in the proliferating human colon CCD-18Co cells when compared with quiescent ones. We also observed ataxia telangiectasia-mutated (ATM) phosphorylation in the quiescent cells. The BITC-induced p53 phosphorylation was counteracted by caffeine treatment, implying the involvement of an ATM/ataxia telangiectasia and Rad3-related kinase signaling pathway. Moreover, downregulation of p53 by a siRNA resulted in the enhancement of susceptibility to undergo apoptosis by BITC. We also showed here that depletion of p53 abrogated G(0)/G(1) arrest accompanied by the declined expression of p21(waf1/cip1) and p27(kip1) in CCD-18Co cells. In conclusion, we identified p53 as a potential negative regulator of the apoptosis induction by BITC in the normal colon CCD-18Co cells through the inhibition of cell-cycle progression at the G(0)/G(1) phase. PMID:17096346

  14. An in silico approach reveals associations between genetic and epigenetic factors within regulatory elements in B cells from primary Sjögren’s syndrome patients

    Directory of Open Access Journals (Sweden)

    Orsia D. Konsta

    2015-08-01

    Full Text Available Recent advances in genetics have highlighted several regions and candidate genes associated with primary Sjögren's syndrome (SS, a systemic autoimmune epithelitis that combines exocrine gland dysfunctions, and focal lymphocytic infiltrations. In addition to genetic factors, it is now clear that epigenetic deregulations are present during SS and restricted to specific cell type subsets such as lymphocytes and salivary gland epithelial cells. In this study, 72 single nucleotide polymorphisms (SNPs associated with 43 SS gene risk factors were selected from publicly available and peer reviewed literature for further in silico analysis. SS risk variant location was tested revealing a broad distribution in coding sequences (5.6%, intronic sequences (55.6%, upstream/downstream genic regions (30.5%, and intergenic regions (8.3%. Moreover, a significant enrichment of regulatory motifs (promoter, enhancer, insulator, DNAse peak and eQTL characterizes SS risk variants (94.4%. Next, screening SNPs in high linkage disequilibrium (r2 ≥ 0.8 in Caucasians revealed 645 new variants including 5 SNPs with missense mutations, and indicated an enrichment of transcriptionally active motifs according to the cell type (B cells > monocytes > T cells >> A549. Finally, we looked at SS risk variants for histone markers in B cells (GM12878, monocytes (CD14+ and epithelial cells (A548. Active histone markers were associated with SS risk variants at both promoters and enhancers in B cells, and within enhancers in monocytes. In conclusion and based on the obtained in silico results, that need further confirmation, associations were observed between SS genetic risk factors and epigenetic factors and these associations predominate in B cells such as those observed at the FAM167A-BLK locus.

  15. Selectable recombinant herpesvirus saimiri is capable of persisting in a human T-cell line.

    OpenAIRE

    Grassmann, R; Fleckenstein, B

    1989-01-01

    Strategies were developed to insert the neo resistance marker into the junction between L DNA and the right terminal repetitive H DNA of herpesvirus saimiri. Recombinant viruses were selectable in permissive epithelioid cultures. The human T-cell line Jurkat could be infected persistently with the Neor virus; the cells contained episomal viral DNA in high copy number. This selectable vector should be generally applicable for gene expression in human T cells.

  16. Involvement of Tspan8 in exosome assembly and target cell selection

    OpenAIRE

    Rana, Sanyukta

    2010-01-01

    Exosomes are the most important intercellular communicators. Tetraspanins/their complexes are suggested to be important in exosomal target cell selection. I showed: changes in Tetraspanin8 associations created from internalization persist upto exosomes and, differences in tetraspanin-complexes on exosomes allow for target cell selectivity.Based on the tetraspanin-complex on exosomes, predictions on potential target cells might be possible, allowing tailored exosome generation for drug delivery.

  17. Redundant transfer of F' plasmids occurs between Escherichia coli cells during nonlethal selections.

    OpenAIRE

    Peters, J E; Benson, S A

    1995-01-01

    Surface exclusion is the mechanism by which F plasmids prevent the redundant entry of additional F plasmids into the host cell during exponential growth. This mechanism is relaxed in cells that are in stationary phase. Using genetically marked F' plasmids and host strains, we extend this finding to Escherichia coli populations during extended nonlethal selection in bacterial lawns. We show that a high level of redundant transfer occurs between these nongrowing cells during the selection. This...

  18. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle signatures in joint diseases.

    Directory of Open Access Journals (Sweden)

    Bence György

    Full Text Available INTRODUCTION: Microvesicles (MVs, earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF samples of patients with osteoarthritis (OA, rheumatoid arthritis (RA and juvenile idiopathic arthritis (JIA. To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM, Nanoparticle Tracking Analysis (NTA and mass spectrometry (MS. For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+ and CD8(+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections. In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction. CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

  19. Genome-wide candidate regions for selective sweeps revealed through massive parallel sequencing of DNA across ten turkey populations

    OpenAIRE

    Aslam, M.L.; Bastiaansen, J. W. M.; Megens, H.J.W.C.; Crooijmans, R.P.M.A.; Blomberg, L.; Groenen, M.

    2014-01-01

    Background The domestic turkey (Meleagris gallopavo) is an important agricultural species that is largely used as a meat-type bird. Characterizing genetic variation in populations of domesticated species and associating these variation patterns with the evolution, domestication, and selective breeding is critical for understanding the dynamics of genomic change in these species. Intense selective breeding and population bottlenecks are expected to leave signatures in the genome of domesticate...

  20. Contrasting Epidemic Histories Reveal Pathogen-Mediated Balancing Selection on Class II MHC Diversity in a Wild Songbird

    OpenAIRE

    Hawley, Dana M; Fleischer, Robert C

    2012-01-01

    The extent to which pathogens maintain the extraordinary polymorphism at vertebrate Major Histocompatibility Complex (MHC) genes via balancing selection has intrigued evolutionary biologists for over half a century, but direct tests remain challenging. Here we examine whether a well-characterized epidemic of Mycoplasmal conjunctivitis resulted in balancing selection on class II MHC in a wild songbird host, the house finch (Carpodacus mexicanus). First, we confirmed the potential for pathogen-...

  1. The metabolic enzyme ManA reveals a link between cell wall integrity and chromosome morphology.

    OpenAIRE

    Maya Elbaz; Sigal Ben-Yehuda

    2010-01-01

    Author Summary The bacterial cell is resistant to extremes of osmotic pressure and protected against mechanical damages by the existence of a rigid outer shell defined as the cell wall. The strength of the cell wall is achieved by the presence of long glycan strands cross-linked by peptide side bridges. The cell wall is a dynamic structure continuously being synthesized and modified to allow for cell growth and division. Damaging the cell wall leads to abnormal cellular morphologies and cell ...

  2. Genetic diversity and parentage in farmer selections of cacao from Southern Sulawesi, Indonesia revealed by microsatellite markers.

    Science.gov (United States)

    Dinarti, Diny; Susilo, Agung W; Meinhardt, Lyndel W; Ji, Kun; Motilal, Lambert A; Mischke, Sue; Zhang, Dapeng

    2015-12-01

    Indonesia is the third largest cocoa-producing country in the world. Knowledge of genetic diversity and parentage of farmer selections is important for effective selection and rational deployment of superior cacao clones in farmers' fields. We assessed genetic diversity and parentage of 53 farmer selections of cacao in Sulawesi, Indonesia, using 152 international clones as references. Cluster analysis, based on 15 microsatellite markers, showed that these Sulawesi farmer selections are mainly comprised of hybrids derived from Trinitario and two Upper Amazon Forastero groups. Bayesian assignment and likelihood-based parentage analysis further demonstrated that only a small number of germplasm groups, dominantly Trinitario and Parinari, contributed to these farmer selections, in spite of diverse parental clones having been used in the breeding program and seed gardens in Indonesia since the 1950s. The narrow parentage predicts a less durable host resistance to cacao diseases. Limited access of the farmers to diverse planting materials or the strong preference for large pods and large bean size by local farmers, may have affected the selection outcome. Diverse sources of resistance, harbored in different cacao germplasm groups, need to be effectively incorporated to broaden the on-farm diversity and ensure sustainable cacao production in Sulawesi. PMID:26719747

  3. Resonant tunneling diodes as energy-selective contacts used in hot-carrier solar cells

    Science.gov (United States)

    Takeda, Yasuhiko; Ichiki, Akihisa; Kusano, Yuya; Sugimoto, Noriaki; Motohiro, Tomoyoshi

    2015-09-01

    Among the four features unique to hot-carrier solar cells (HC-SCs): (i) carrier thermalization time and (ii) carrier equilibration time in the absorber, (iii) energy-selection width and (iv) conductance of the energy-selective contacts (ESCs), requisites of (i)-(iii) for high conversion efficiency have been clarified. We have tackled the remaining issues related to (iv) in the present study. The detailed balance model of HC-SC operation has been improved to involve a finite value of the ESC conductance to find the required values, which in turn has been revealed to be feasible using resonant tunneling diodes (RTDs) consisting of semiconductor quantum dots (QDs) and quantum wells (QWs) by means of a formulation to calculate the conductance of the QD- and QW-RTDs derived using the rigorous solutions of the effective-mass Hamiltonians. Thus, all of the four requisites unique to HC-SCs to achieve high conversion efficiency have been elucidated, and the two requisites related to the ESCs can be fulfilled using the QD- and QW-RTDs.

  4. Resonant tunneling diodes as energy-selective contacts used in hot-carrier solar cells

    International Nuclear Information System (INIS)

    Among the four features unique to hot-carrier solar cells (HC-SCs): (i) carrier thermalization time and (ii) carrier equilibration time in the absorber, (iii) energy-selection width and (iv) conductance of the energy-selective contacts (ESCs), requisites of (i)-(iii) for high conversion efficiency have been clarified. We have tackled the remaining issues related to (iv) in the present study. The detailed balance model of HC-SC operation has been improved to involve a finite value of the ESC conductance to find the required values, which in turn has been revealed to be feasible using resonant tunneling diodes (RTDs) consisting of semiconductor quantum dots (QDs) and quantum wells (QWs) by means of a formulation to calculate the conductance of the QD- and QW-RTDs derived using the rigorous solutions of the effective-mass Hamiltonians. Thus, all of the four requisites unique to HC-SCs to achieve high conversion efficiency have been elucidated, and the two requisites related to the ESCs can be fulfilled using the QD- and QW-RTDs

  5. Study on sickle cell disease haplotypes reveals the African origin of Amapá's population

    Directory of Open Access Journals (Sweden)

    Natália de Morais Castelo

    2014-04-01

    Full Text Available Introduction:Sickle cell disease (SCD is a hereditary, hematologic, multifactorial disease, with high prevalence worldwide; its cause is a mutation in the sixth codon of the beta globin gene (βs.Objective:To identify the haplotypes present in people with SCD in Amapá, and relate them to African descent.Methods:We analyzed, by molecular techniques, 46 blood samples from people with SCD in Macapá, the capital of Amapá, with the purpose of obtaining information about haplotype frequency distribution, which helps understand the ethnic background of Amapá's population.Results:Our study revealed that the most frequent haplotype is Bantu (61.2%, followed by Benin (26.6% and Senegal (12.2%. Results showed statistical differences from studies conducted in other regions. A high frequency of the Senegal haplotype stands out, in comparison with some Brazilian studies.Conclusion:Amapá's results exhibit unique characteristics when compared to haplotypes in other regions, with high frequency of Senegal and Benin haplotypes, absence of atypical, Cameroon and Saudi, confirming that Brazil shows ethnic background diversity, as well as different haplotype frequencies.

  6. Single-cell genomics reveal metabolic strategies for microbial growth and survival in an oligotrophic aquifer

    Energy Technology Data Exchange (ETDEWEB)

    Wilkins, Michael J.; Kennedy, David W.; Castelle, Cindy; Field, Erin; Stepanauskas, Ramunas; Fredrickson, Jim K.; Konopka, Allan

    2014-02-09

    Bacteria from the genus Pedobacter are a major component of microbial assemblages at Hanford Site and have been shown to significantly change in abundance in response to the subsurface intrusion of Columbia River water. Here we employed single cell genomics techniques to shed light on the physiological niche of these microorganisms. Analysis of four Pedobacter single amplified genomes (SAGs) from Hanford Site sediments revealed a chemoheterotrophic lifestyle, with the potential to exist under both aerobic and microaerophilic conditions via expression of both aa3­-type and cbb3-type cytochrome c oxidases. These SAGs encoded a wide-range of both intra-and extra­-cellular carbohydrate-active enzymes, potentially enabling the degradation of recalcitrant substrates such as xylan and chitin, and the utilization of more labile sugars such as mannose and fucose. Coupled to these enzymes, a diversity of transporters and sugar-binding molecules were involved in the uptake of carbon from the extracellular local environment. The SAGs were enriched in TonB-dependent receptors (TBDRs), which play a key role in uptake of substrates resulting from degradation of recalcitrant carbon. CRISPR-Cas mechanisms for resisting viral infections were identified in all SAGs. These data demonstrate the potential mechanisms utilized for persistence by heterotrophic microorganisms in a carbon-limited aquifer, and hint at potential linkages between observed Pedobacter abundance shifts within the 300 Area subsurface and biogeochemical shifts associated with Columbia River water intrusion.

  7. Pushing structural limits to reveal fundamental mechanisms of organic solar cell operation

    Science.gov (United States)

    Rand, Barry

    2015-03-01

    Organic-based solar cells are beginning to emerge with the potential to compete with other thin film photovoltaic technologies, with efficiencies of 12% recently demonstrated. Unique to the function of organic photovoltaics are the creation of tightly bound excitons that can only be efficiently separated at a donor/acceptor (D/A) interface capable of providing the necessary energetic driving force for dissociation. The consequences of this are the need for long exciton diffusion lengths and the presence of charge transfer (CT) states, ground state complexes that exist at the D/A interface. We have found that charge transfer states are more easily separated into free charge if they are delocalized; an aspect that becomes most feasible for highly ordered systems. I will discuss our recent efforts to template and control film morphology and molecular orientation. These studies allow us to understand the importance of molecular orientation, crystallite size, and crystal phase. We will show templated devices utilizing neat films as well as bulk heterojunctions, with crystallite dimensions spanning from the more standard nano-sized grains to those with highly ordered micron-sized crystalline domains revealing unprecedented thin film exciton diffusion lengths of 100s of nm. In these highly ordered films, owing to significant delocalization, we are able to directly measure photocurrent from multiple CT states, an aspect which has important consequences for the design of more efficient photocurrent generation. We acknowledge support from DOE BES Grant #11493344.

  8. Piperlongumine selectively kills cancer cells and increases cisplatin antitumor activity in head and neck cancer.

    Science.gov (United States)

    Roh, Jong-Lyel; Kim, Eun Hye; Park, Jin Young; Kim, Ji Won; Kwon, Minsu; Lee, Byung-Heon

    2014-10-15

    Adaptation to cellular stress is not a vital function of normal cells but is required of cancer cells, and as such might be a sensible target in cancer therapy. Piperlongumine is a naturally occurring small molecule selectively toxic to cancer cells. This study assesses the cytotoxicity of piperlongumine and its combination with cisplatin in head-and-neck cancer (HNC) cells in vitro and in vivo. The effect of piperlongumine, alone and in combination with cisplatin, was assessed in human HNC cells and normal cells by measuring growth, death, cell cycle progression, reactive oxygen species (ROS) production, and protein expression, and in tumor xenograft mouse models. Piperlongumine killed HNC cells regardless of p53 mutational status but spared normal cells. It increased ROS accumulation in HNC cells, an effect that can be blocked by the antioxidant N-acetyl-L-cysteine. Piperlongumine induced selective cell death in HNC cells by targeting the stress response to ROS, leading to the induction of death pathways involving JNK and PARP. Piperlongumine increased cisplatin-induced cytotoxicity in HNC cells in a synergistic manner in vitro and in vivo. Piperlongumine might be a promising small molecule with which to selectively kill HNC cells and increase cisplatin antitumor activity by targeting the oxidative stress response. PMID:25193861

  9. Representation of spontaneous movement by dopaminergic neurons is cell-type selective and disrupted in parkinsonism.

    Science.gov (United States)

    Dodson, Paul D; Dreyer, Jakob K; Jennings, Katie A; Syed, Emilie C J; Wade-Martins, Richard; Cragg, Stephanie J; Bolam, J Paul; Magill, Peter J

    2016-04-12

    Midbrain dopaminergic neurons are essential for appropriate voluntary movement, as epitomized by the cardinal motor impairments arising in Parkinson's disease. Understanding the basis of such motor control requires understanding how the firing of different types of dopaminergic neuron relates to movement and how this activity is deciphered in target structures such as the striatum. By recording and labeling individual neurons in behaving mice, we show that the representation of brief spontaneous movements in the firing of identified midbrain dopaminergic neurons is cell-type selective. Most dopaminergic neurons in the substantia nigra pars compacta (SNc), but not in ventral tegmental area or substantia nigra pars lateralis, consistently represented the onset of spontaneous movements with a pause in their firing. Computational modeling revealed that the movement-related firing of these dopaminergic neurons can manifest as rapid and robust fluctuations in striatal dopamine concentration and receptor activity. The exact nature of the movement-related signaling in the striatum depended on the type of dopaminergic neuron providing inputs, the striatal region innervated, and the type of dopamine receptor expressed by striatal neurons. Importantly, in aged mice harboring a genetic burden relevant for human Parkinson's disease, the precise movement-related firing of SNc dopaminergic neurons and the resultant striatal dopamine signaling were lost. These data show that distinct dopaminergic cell types differentially encode spontaneous movement and elucidate how dysregulation of their firing in early Parkinsonism can impair their effector circuits. PMID:27001837

  10. Cytokine-Regulated GADD45G Induces Differentiation and Lineage Selection in Hematopoietic Stem Cells

    Directory of Open Access Journals (Sweden)

    Frederic B. Thalheimer

    2014-07-01

    Full Text Available The balance of self-renewal and differentiation in long-term repopulating hematopoietic stem cells (LT-HSC must be strictly controlled to maintain blood homeostasis and to prevent leukemogenesis. Hematopoietic cytokines can induce differentiation in LT-HSCs; however, the molecular mechanism orchestrating this delicate balance requires further elucidation. We identified the tumor suppressor GADD45G as an instructor of LT-HSC differentiation under the control of differentiation-promoting cytokine receptor signaling. GADD45G immediately induces and accelerates differentiation in LT-HSCs and overrides the self-renewal program by specifically activating MAP3K4-mediated MAPK p38. Conversely, the absence of GADD45G enhances the self-renewal potential of LT-HSCs. Videomicroscopy-based tracking of single LT-HSCs revealed that, once GADD45G is expressed, the development of LT-HSCs into lineage-committed progeny occurred within 36 hr and uncovered a selective lineage choice with a severe reduction in megakaryocytic-erythroid cells. Here, we report an unrecognized role of GADD45G as a central molecular linker of extrinsic cytokine differentiation and lineage choice control in hematopoiesis.

  11. Low dose irradiation of thyroid cells reveals a unique transcriptomic and epigenetic signature in RET/PTC-positive cells

    Energy Technology Data Exchange (ETDEWEB)

    Abou-El-Ardat, Khalil, E-mail: kabouela@sckcen.be [Radiobiology Unit, Molecular and Cellular Biology, GKD Building, Studiecentrum voor Kernenergie - Centre d' Etude de l' Energie Nucleaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); Monsieurs, Pieter [Radiobiology Unit, Molecular and Cellular Biology, GKD Building, Studiecentrum voor Kernenergie - Centre d' Etude de l' Energie Nucleaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); Anastasov, Natasa; Atkinson, Mike [Department of Radiation Sciences, Helmholtz Zentrum Muenchen, Munich (Germany); Derradji, Hanane [Radiobiology Unit, Molecular and Cellular Biology, GKD Building, Studiecentrum voor Kernenergie - Centre d' Etude de l' Energie Nucleaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); De Meyer, Tim [Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); Department of Applied Mathematics, Biometrics and Process Control, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); Bekaert, Sofie [Clinical Research Center, Faculty for Medicine and Health Sciences, Universiteit Gent, 185 De Pintelaan, 9000 Ghent (Belgium); Van Criekinge, Wim [Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); and others

    2012-03-01

    The high doses of radiation received in the wake of the Chernobyl incident and the atomic bombing of Hiroshima and Nagasaki have been linked to the increased appearance of thyroid cancer in the children living in the vicinity of the site. However, the data gathered on the effect of low doses of radiation on the thyroid remain limited. We have examined the genome wide transcriptional response of a culture of TPC-1 human cell line of papillary thyroid carcinoma origin with a RET/PTC1 translocation to various doses (0.0625, 0.5, and 4 Gy) of X-rays and compared it to response of thyroids with a RET/PTC3 translocation and against wild-type mouse thyroids irradiated with the same doses using Affymetrix microarrays. We have found considerable overlap at a high dose of 4 Gy in both RET/PTC-positive systems but no common genes at 62.5 mGy. In addition, the response of RET/PTC-positive system at all doses was distinct from the response of wild-type thyroids with both systems signaling down different pathways. Analysis of the response of microRNAs in TPC-1 cells revealed a radiation-responsive signature of microRNAs in addition to dose-responsive microRNAs. Our results point to the fact that a low dose of X-rays seems to have a significant proliferative effect on normal thyroids. This observation should be studied further as opposed to its effect on RET/PTC-positive thyroids which was subtle, anti-proliferative and system-dependent.

  12. Low dose irradiation of thyroid cells reveals a unique transcriptomic and epigenetic signature in RET/PTC-positive cells.

    Science.gov (United States)

    Abou-El-Ardat, Khalil; Monsieurs, Pieter; Anastasov, Nataša; Atkinson, Mike; Derradji, Hanane; De Meyer, Tim; Bekaert, Sofie; Van Criekinge, Wim; Baatout, Sarah

    2012-03-01

    The high doses of radiation received in the wake of the Chernobyl incident and the atomic bombing of Hiroshima and Nagasaki have been linked to the increased appearance of thyroid cancer in the children living in the vicinity of the site. However, the data gathered on the effect of low doses of radiation on the thyroid remain limited. We have examined the genome wide transcriptional response of a culture of TPC-1 human cell line of papillary thyroid carcinoma origin with a RET/PTC1 translocation to various doses (0.0625, 0.5, and 4Gy) of X-rays and compared it to response of thyroids with a RET/PTC3 translocation and against wild-type mouse thyroids irradiated with the same doses using Affymetrix microarrays. We have found considerable overlap at a high dose of 4Gy in both RET/PTC-positive systems but no common genes at 62.5mGy. In addition, the response of RET/PTC-positive system at all doses was distinct from the response of wild-type thyroids with both systems signaling down different pathways. Analysis of the response of microRNAs in TPC-1 cells revealed a radiation-responsive signature of microRNAs in addition to dose-responsive microRNAs. Our results point to the fact that a low dose of X-rays seems to have a significant proliferative effect on normal thyroids. This observation should be studied further as opposed to its effect on RET/PTC-positive thyroids which was subtle, anti-proliferative and system-dependent. PMID:22027090

  13. Double suicide genes selectively kill human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Liu Lunxu

    2011-02-01

    Full Text Available Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli cytosine deaminase (CD gene and the herpes simplex virus-thymidine kinase (TK gene were cloned using polymerase chain reaction (PCR. Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304 and KDR-negative liver cancer cell line (HepG2 were infected with the recombinant adenoviruses at different multiplicity of infection (MOI. The infection rate was measured by green fluorescent protein (GFP expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV and/or 5-fluorocytosine (5-FC. The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL staining. Results Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs. Conclusion AdKDR-CDglyTK/double prodrog system may be a useful

  14. Selective stimulation of prostatic carcinoma cell proliferation by transferrin.

    OpenAIRE

    M.C. Rossi; Zetter, B R

    1992-01-01

    Aggressive prostatic carcinomas most frequently metastasize to the skeletal system. We have previously shown that cultured human prostatic carcinoma cells are highly responsive to growth factors found in human bone marrow. To identify the factor(s) responsible for the increased prostatic carcinoma cell proliferation, we fractionated crude bone marrow preparations by using hydroxylapatite HPLC. The major activity peak contained two high molecular weight bands (M(r) = 80,000 and 69,000) that cr...

  15. Crystal Structure of a Thermostable Alanine Racemase from Thermoanaerobacter tengcongensis MB4 Reveals the Role of Gln360 in Substrate Selection

    Science.gov (United States)

    Wang, Xiaoyan; Xu, Shujing; Ju, Jiansong; Xu, Xiaoling

    2015-01-01

    Pyridoxal 5’-phosphate (PLP) dependent alanine racemase catalyzes racemization of L-Ala to D-Ala, a key component of the peptidoglycan network in bacterial cell wall. It has been extensively studied as an important antimicrobial drug target due to its restriction in eukaryotes. However, many marketed alanine racemase inhibitors also act on eukaryotic PLP-dependent enzymes and cause side effects. A thermostable alanine racemase (AlrTt) from Thermoanaerobacter tengcongensis MB4 contains an evolutionarily non-conserved residue Gln360 in inner layer of the substrate entryway, which is supposed to be a key determinant in substrate specificity. Here we determined the crystal structure of AlrTt in complex with L-Ala at 2.7 Å resolution, and investigated the role of Gln360 by saturation mutagenesis and kinetic analysis. Compared to typical bacterial alanine racemase, presence of Gln360 and conformational changes of active site residues disrupted the hydrogen bonding interactions necessary for proper PLP immobilization, and decreased both the substrate affinity and turnover number of AlrTt. However, it could be complemented by introduction of hydrophobic amino acids at Gln360, through steric blocking and interactions with a hydrophobic patch near active site pocket. These observations explained the low racemase activity of AlrTt, revealed the essential role of Gln360 in substrate selection, and its preference for hydrophobic amino acids especially Tyr in bacterial alanine racemization. Our work will contribute new insights into the alanine racemization mechanism for antimicrobial drug development. PMID:26218070

  16. A selective screening platform reveals unique global expression patterns of microRNAs in a cohort of human soft-tissue sarcomas.

    Science.gov (United States)

    Yu, Peter Y; Balkhi, Mumtaz Y; Ladner, Katherine J; Alder, Hansjuerg; Yu, Lianbo; Mo, Xiaokui; Kraybill, William G; Guttridge, Denis C; Hans Iwenofu, O

    2016-04-01

    Sarcomas are malignant heterogenous tumors of mesenchymal derivation. Emerging data suggest that miRNA might have a causal role in sarcomagenesis. Herein, we used a selective miRNA screening platform to study the comparative global miRNA expression signatures in a cohort of human sarcomas with the caveat that comparisons between tumor and non-tumor cells were performed from the same patients using formalin-fixed paraffin-embedded tissue. Five histologic types were examined that included: myxoid liposarcoma, well-differentiated liposarcoma, dedifferentiated liposarcoma, pleomorphic rhabdomyosarcoma, and synovial sarcoma. In addition, soft-tissue lipomas and normal fat were included as a separate set of controls for the lipogenic tumors. Clustering analysis showed a distinct global difference in expression patterns between the normal and sarcoma tissues. Expression signatures in an unsupervised hierarchical clustering analysis revealed tight clustering in synovial and myxoid liposarcomas, and the least clustering was observed in the pleomorphic rhabdomyosarcoma subtype. MiR-145 showed underexpression in pleomorphic rhabdomyosarcoma, well-differentiated liposarcoma, and synovial sarcoma. Unexpectedly, we found that a set of muscle-specific microRNAs (miRNAs; myomiRs): miR-133, miR-1, and miR-206 was significantly underexpressed in well-differentiated liposarcoma and synovial sarcoma, suggesting that they may function as tumor suppressors as described in muscle-relevant rhabdomyosarcomas. In addition, a tight linear progression of miRNA expression was identified from normal fat to dedifferentiated liposarcoma. These results suggest that miRNA expression profiles could elucidate classes of miRNAs that may elicit tumor-relevant activities in specific sarcoma subtypes. PMID:26878133

  17. Side Population Cells from Human Melanoma Tumors Reveal Diverse Mechanisms for Chemoresistance

    OpenAIRE

    Luo, Yuchun; Ellis, Lixia Z; Dallaglio, Katiuscia; Takeda, Moe; Robinson, William A.; Robinson, Steven; Liu, Weimin; Lewis, Karl D.; McCarter, Martin D; Gonzalez, Rene; David A Norris; Roop, Dennis R.; Spritz, Richard A.; Ahn, Natalie G.; Fujita, Mayumi

    2012-01-01

    Side population (SP) is identified as cells capable of excluding the fluorescent Hoechst dye and anticancer drugs, and represents hematopoietic stem cells and chemoresistant cells from several solid tumors. In this study, we confirmed the presence of SP cells in tumors from melanoma patients. Melanoma SP cells overexpressed ATP-binding-cassette (ABC) transporters, ABCB1 and ABCB5. We generated a direct in vivo xenograft model, and demonstrated that SP cells were resistant to paclitaxel, a sub...

  18. Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity.

    Science.gov (United States)

    Markovski, Monica; Bohrhunter, Jessica L; Lupoli, Tania J; Uehara, Tsuyoshi; Walker, Suzanne; Kahne, Daniel E; Bernhardt, Thomas G

    2016-04-26

    To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a of Escherichia coli require the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b-LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed. PMID:27071112

  19. A selective tropism of transfused oval cells for liver

    Institute of Scientific and Technical Information of China (English)

    Jian-Zhi Chen; Hai Hong; Jin Xiang; Ling Xue; Guo-Qiang Zhao

    2003-01-01

    AIM: To explore the biological behaviors of hepatic oval cells after transfused into the circulation of experimental animals.METHODS: Oval cells from male SD rat were transfused into the circulation of a female rat which were treated by a 2-AAF/CCl4 program, through caudal vein. Sex-determining gene sry which located on Y chromosome was examined by PCR and in situ hybridization technique in liver, kidney and spleen of the experimental animals, respectively.RESULTS: The results of the cell-transplant experiment showed that the srygene was detectable only in the liver but not in spleen and kidney of the experimental rats, and no signals could be detected in the control animals. It can be also morphologically proved that some exogenous cells had migrated into the parenchyma of the liver and settled there.CONCLUSION: The result means that there are exogenous cells located in the liver of the experimental animal and the localization is specific to the liver. This indicates that some "signal molecules" must exist in the circulation of the rats treated by 2-AAF/CCl4. These "signal molecules" might play an important role in specific localization and differentiation of transfused oval cells.

  20. Species-scanning mutagenesis of the serotonin transporter reveals residues essential in selective, high-affinity recognition of antidepressants

    DEFF Research Database (Denmark)

    Mortensen, O V; Kristensen, A S; Wiborg, O

    2001-01-01

    The serotonin transporter (SERT) is a high-affinity sodium/chloride-dependent neurotransmitter transporter responsible for reuptake of serotonin from the extracellular space. SERT is a selective target of several clinically important antidepressants. In a cross-species analysis comparing human and...... antidepressants citalopram, fluoxetine, paroxetine and imipramine were several-fold higher at hSERT compared with bSERT. No species selectivity was observed for the antidepressants fluvoxamine, and sertraline or for the psychostimulants cocaine, the cocaine analogue beta-carbomethoxy-3beta-(4-iodophenyl...

  1. Biomarker screening of oral cancer cell lines revealed sub-populations of CD133-, CD44-, CD24- and ALDH1- positive cancer stem cells

    Directory of Open Access Journals (Sweden)

    Kendall K

    2013-05-01

    Full Text Available Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for cancer-related mortality. For the past several decades the mainstay of treatment for HNSCC has been surgery and external beam radiation, although more recent trials combining chemotherapy and radiation have demonstrated improvements. However, cancer recurrence and treatment failures continue to occur in a significant percentage of patients. Recent advances in tumor biology have led to the discovery that many cancers, including HNSCC, may contain subpopulations of cells with stem cell-like properties that may explain relapse and recurrence. The objective of this study was to screen existing oral cancer cell lines for biomarkers specific for cells with stem cell-like properties. RNA was isolated for RT-PCR screening using primers for specific mRNA of the biomarkers: CD44, CD24, CD133, NANOG, Nestin, ALDH1, and ABCG2 in CAL27, SCC25 and SCC15 cells. This analysis revealed that some oral cancer cell lines (CAL27 and SCC25 may contain small subpopulations of adhesion- and contact-independent cells (AiDC that also express tumor stem cell markers, including CD44, CD133, and CD24. In addition, CAL27 cells also expressed the intracellular tumor stem cell markers, ALDH1 and ABCG2. Isolation and culture of the adhesion- and contact-independent cells from CAL27 and SCC25 populations revealed differential proliferation rates and more robust inhibition by the MEK inhibitor PD98059, as well as the chemotherapeutic agents Cisplatin and Paclitaxel, within the AiDC CAL27 cells. At least one oral cancer cell line (CAL27 contained subpopulations of cells that express specific biomarkers associated with tumor stem cells which were morphologically and phenotypically distinct from other cells within this cell line.

  2. In Vitro Selection of Cancer Cell-Specific Molecular Recognition Elements from Amino Acid Libraries

    Science.gov (United States)

    Williams, Ryan M.; Sooter, Letha J.

    2015-01-01

    Differential cell systematic evolution of ligands by exponential enrichment (SELEX) is an in vitro selection method for obtaining molecular recognition elements (MREs) that specifically bind to individual cell types with high affinity. MREs are selected from initial large libraries of different nucleic or amino acids. This review outlines the construction of peptide and antibody fragment libraries as well as their different host types. Common methods of selection are also reviewed. Additionally, examples of cancer cell MREs are discussed, as well as their potential applications. PMID:26436100

  3. Circulating cancer stem cells: the importance to select.

    Science.gov (United States)

    Yang, Ming-Hsin; Imrali, Ahmet; Heeschen, Christopher

    2015-10-01

    It has been demonstrated that even localized tumors without clinically apparent metastasis give rise to circulating tumor cells (CTCs). A growing number of technically diverse platforms are being developed for detecting/isolating CTCs in the circulating blood. Despite the technical challenges of isolating rare CTCs from blood, recent studies have already shown the predictive value of CTCs enumeration. Thus, it is becoming increasingly accepted that CTC numbers are linked to patients' outcome and may also be used to monitor treatment response and disease relapse, respectively. Further CTCs provide a non-invasive source for tumor material, 'liquid biopsy', which is particularly important for patients, where no biopsy material can be obtained or where serial biopsies of the tumor, e.g., following treatment, are practically impossible. On the other hand the molecular and biological characterization of CTCs has still remained at a rather experimental stage. Future studies are necessary to define CTC heterogeneity to establish the crucial role of circulating cancer stem cells for driving metastasis, which represent a distinct subpopulation of CTCs that bear metastasis-initiating capabilities based on their stemness properties and invasiveness and thus are critical for the patients' clinical outcome. As compared to non-tumorigenic/metastatic bulk CTCs, circulating cancer stem cells may not only be capable of evading from the primary tumor, but also escape from immune surveillance, survive in the circulating blood and subsequently form metastases in distant organs. Thus, circulating cancer stem cells represent a subset of exclusively tumorigenic cancer stem cells characterized by their invasive characteristics and are potential therapeutic targets for preventing disease progression. To date, only a few original reports and reviews have been published focusing on circulating cancer stem cells. This review discusses the potential importance of isolating and characterizing

  4. A Nanoprinted Model of Interstitial Cancer Migration Reveals a Link between Cell Deformability and Proliferation.

    Science.gov (United States)

    Panagiotakopoulou, Magdalini; Bergert, Martin; Taubenberger, Anna; Guck, Jochen; Poulikakos, Dimos; Ferrari, Aldo

    2016-07-26

    Metastatic progression of tumors requires the coordinated dissemination of cancerous cells through interstitial tissues and their replication in distant body locations. Despite their importance in cancer treatment decisions, key factors, such as cell shape adaptation and the role it plays in dense tissue invasion by cancerous cells, are not well understood. Here, we employ a 3D electrohydrodynamic nanoprinting technology to generate vertical arrays of topographical pores that mimic interstitial tissue resistance to the mesenchymal migration of cancerous cells, in order to determine the effect of nuclear size, cell deformability, and cell-to-substrate adhesion on tissue invasion efficiency. The high spatial and temporal resolution of our analysis demonstrates that the ability of cells to deform depends on the cell cycle phase, peaks immediately after mitosis, and is key to the invasion process. Increased pore penetration efficiency by cells in early G1 phase also coincided with their lower nuclear volume and higher cell deformability, compared with the later cell cycle stages. Furthermore, artificial decondensation of chromatin induced an increase in cell and nuclear deformability and improved pore penetration efficiency of cells in G1. Together, these results underline that along the cell cycle cells have different abilities to dynamically remodel their actin cytoskeleton and induce nuclear shape changes, which determines their pore penetration efficiency. Thus, our results support a mechanism in which cell proliferation and pore penetration are functionally linked to favor the interstitial dissemination of metastatic cells. PMID:27268411

  5. Microevolution in time and space: SNP analysis of historical DNA reveals dynamic signatures of selection in Atlantic cod

    DEFF Research Database (Denmark)

    Therkildsen, Nina Overgaard; Hansen, Jakob Hemmer; Als, Thomas Damm;

    2013-01-01

    Little is known about how quickly natural populations adapt to changes in their environment and how temporal and spatial variation in selection pressures interact to shape patterns of genetic diversity. We here address these issues with a series of genome scans in four overfished populations...

  6. Prediction of cell-penetrating peptides with feature selection techniques.

    Science.gov (United States)

    Tang, Hua; Su, Zhen-Dong; Wei, Huan-Huan; Chen, Wei; Lin, Hao

    2016-08-12

    Cell-penetrating peptides are a group of peptides which can transport different types of cargo molecules such as drugs across plasma membrane and have been applied in the treatment of various diseases. Thus, the accurate prediction of cell-penetrating peptides with bioinformatics methods will accelerate the development of drug delivery systems. The study aims to develop a powerful model to accurately identify cell-penetrating peptides. At first, the peptides were translated into a set of vectors with the same dimension by using dipeptide compositions. Secondly, the Analysis of Variance-based technique was used to reduce the dimension of the vector and explore the optimized features. Finally, the support vector machine was utilized to discriminate cell-penetrating peptides from non-cell-penetrating peptides. The five-fold cross-validated results showed that our proposed method could achieve an overall prediction accuracy of 83.6%. Based on the proposed model, we constructed a free webserver called C2Pred (http://lin.uestc.edu.cn/server/C2Pred). PMID:27291150

  7. Piperlongumine selectively kills cancer cells and increases cisplatin antitumor activity in head and neck cancer

    OpenAIRE

    Roh, Jong-Lyel; Kim, Eun Hye; Park, Jin Young; Kim, Ji Won; Kwon, Minsu; Lee, Byung-Heon

    2014-01-01

    Adaptation to cellular stress is not a vital function of normal cells but is required of cancer cells, and as such might be a sensible target in cancer therapy. Piperlongumine is a naturally occurring small molecule selectively toxic to cancer cells. This study assesses the cytotoxicity of piperlongumine and its combination with cisplatin in head-and-neck cancer (HNC) cells in vitro and in vivo. The effect of piperlongumine, alone and in combination with cisplatin, was assessed in human HNC c...

  8. Selective differentiation and proliferation of hematopoietic cells induced by recombinant human interleukins.

    OpenAIRE

    Saito, H; Hatake, K.; Dvorak, A. M.; Leiferman, K M; Donnenberg, A D; Arai, N.; Ishizaka, K; Ishizaka, T

    1988-01-01

    Effects of recombinant human interleukins on hematopoiesis were explored by using suspension cultures of mononuclear cells of human umbilical-cord blood and bone marrow. The results showed that interleukin 5 induced the selective differentiation and proliferation of eosinophils. After 3 weeks in culture with interleukin 5, essentially all nonadherent cells in both bone marrow and cord blood cell cultures became eosinophilic myelocytes. Culture of the same cells with interleukin 4 resulted in ...

  9. A novel potent Fas agonist for selective depletion of tumor cells in hematopoietic transplants

    OpenAIRE

    Nahimana, A; AUBRY, D.; Lagopoulos, L; Greaney, P.; Attinger, A; Demotz, S; Dawson, K. M.; Schapira, M; Tschopp, J; Dupuis, M.; Duchosal, M A

    2011-01-01

    There remains a clear need for effective tumor cell purging in autologous stem cell transplantation (ASCT) where residual malignant cells within the autograft contribute to disease relapse. Here we propose the use of a novel Fas agonist with potent pro-apoptotic activity, termed MegaFasL, as an effective ex-vivo purging agent. MegaFasL selectively kills hematological cancer cells from lymphomas and leukemias and prevents tumor development at concentrations that do not reduce the functional ca...

  10. Selective tropism of liver stem cells to hepatocellular carcinoma in vivo

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate the selective tropism of liver stem cells to hepatocellular carcinoma (HCC) in an animal model and its feasibility as a vector to deliver therapeutic genes for targeted therapy of HCC.METHODS: WB-F344, a kind of rat liver stem cell,was infected with recombinant virus to establish a cell line with stable, high-level expressing enhanced green fluorescent protein (EGFP). An animal model of HCC in Wistar rats was established by implanting HCC cells (CBRH7919) combined with an immunosuppressive drug.EGFP labeled liver stem cells were injected into caudal veins of the animals and distribution was observed at different time points after injection. SDF-1 and c-kit expression in non-tumor liver and tumor tissue were analysed by immunohistochemistry for the relationshiop between the expression and migration of liver stem cells.Furthermore, hepatic stem cells were injected via the portal vein, hepatic artery, caudal vein, or directly into the pericancerous liver tissue, respectively, and effects on migration, localization, and proliferation of the hepatic stem cells within the tumor tissue were observed and analyzed.RESULTS: Recombinant adenovirus could deliver the EGFP gene to hepatic stem cells. A new stem cell line,named WB-EGFP, was established that stably expressed EGFP. WB-EGFP cells still showed selective tropism towards HCC and EGFP expression was stable in vivo.According to immunohistochemistry results, SDF-1 may not be related to the mechanisms of tropism of hepatic stem cells. Different application sites affected the distribution of liver stem cells. Injection via the portal vein was superior with regard to selective migration,localization, and proliferation of the hepatic stem cells within the tumor tissue.CONCLUSION: Liver stem cells have the biological behavior of selective migration to HCC in vivo and they could localize and proliferate within HCC tissue stably expressing the target gene. Liver stem cells are a potential tool for a targeted

  11. A Systematic Analysis of Cell Cycle Regulators in Yeast Reveals That Most Factors Act Independently of Cell Size to Control Initiation of Division

    OpenAIRE

    Scott A Hoose; Jeremy A Rawlings; Kelly, Michelle M.; M Camille Leitch; Ababneh, Qotaiba O; Robles, Juan P.; David Taylor; Hoover, Evelyn M.; Bethel Hailu; McEnery, Kayla A.; S Sabina Downing; Deepika Kaushal; Yi Chen; Alex Rife; Kirtan A Brahmbhatt

    2012-01-01

    Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene dele...

  12. Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin

    OpenAIRE

    Zhu, Lixin; Hatakeyama, Jason; Zhang, Bing; Makdisi, Joy; Ender, Cody; Forte, John G.

    2008-01-01

    ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an inc...

  13. Intestinal crypt homeostasis revealed at single stem cell level by in vivo live-imaging

    Science.gov (United States)

    Zomer, Anoek; Snippert, Hugo J.; de Sauvage, Frederic J.; Simons, Benjamin D.; Clevers, Hans; van Rheenen, Jacco

    2014-01-01

    Summary The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt1. Alongside Lgr5, intestinal stem cells have been associated with various markers, which are expressed heterogeneously within the crypt base region1-6. Previous quantitative clonal fate analyses have proposed that homeostasis occurs as the consequence of neutral competition between dividing stem cells7-9. However, the short-term behaviour of individual Lgr5+ cells positioned at different locations within the crypt base compartment has not been resolved. Here, we established the short-term dynamics of intestinal stem cells using a novel approach of continuous intravital imaging of Lgr5-Confetti mice. We find that Lgr5+ cells in the upper part of the niche (termed ‘border cells’) can be passively displaced into the transit-amplifying (TA) domain, following division of proximate cells, implying that determination of stem cell fate can be uncoupled from division. Through the quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed ‘central cells’, experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5+ cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem cell maintenance in which a dynamically heterogeneous cell population is able to function long-term as a single stem cell pool. PMID:24531760

  14. Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44-positive cancer cells.

    Science.gov (United States)

    Aires, Antonio; Ocampo, Sandra M; Simões, Bruno M; Josefa Rodríguez, María; Cadenas, Jael F; Couleaud, Pierre; Spence, Katherine; Latorre, Alfonso; Miranda, Rodolfo; Somoza, Álvaro; Clarke, Robert B; Carrascosa, José L; Cortajarena, Aitziber L

    2016-02-12

    Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully  apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro. PMID:26754042

  15. Selective killing of methotrexate-resistant cells carrying amplified dihydrofolate reductase genes

    International Nuclear Information System (INIS)

    A method for the selective killing of methotrexate (MTX)-resistant cells has been developed. The selection is based on the incorporation of tritiated deoxyuridine into the DNA of MTX-resistant cells but not normal MTX-sensitive cells in the presence of the drug. A Chinese hamster ovary cell mutant that overproduces dihydrofolate reductase was used as an example of a MTX-resistant cell line. In this system, a 10,000-fold enrichment for wild-type MTX-sensitive cells could be achieved after 24 hr of exposure to the drug combination. This selection technique was applied to the isolation of MTX-sensitive segregants from hybrid cells formed between the MTX-resistant mutant and wild-type cells. The loss of MTX resistance and dihydrofolate reductase overproduction was always accompanied by the loss of a homogeneously staining region on chromosome 2 of the resistant parent that contains the amplified genes specifying this enzyme. While this region is always lost, other parts of chromosome 2 are almost always retained, suggesting that deletion rather than chromosome loss underlies marker segregation in this case. When the selection was applied to the resistant mutant itself, no MTX-sensitive revertants were obtained among 10(5) cells screened, attesting to the stability of gene amplification in this clone. It is suggested that this combination of drugs may be useful for the elimination of MTX-resistant tumor cells that develop after MTX chemotherapy

  16. Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44-positive cancer cells

    Science.gov (United States)

    Aires, Antonio; Ocampo, Sandra M.; Simões, Bruno M.; Josefa Rodríguez, María; Cadenas, Jael F.; Couleaud, Pierre; Spence, Katherine; Latorre, Alfonso; Miranda, Rodolfo; Somoza, Álvaro; Clarke, Robert B.; Carrascosa, José L.; Cortajarena, Aitziber L.

    2016-02-01

    Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.

  17. An in silico Approach Reveals Associations between Genetic and Epigenetic Factors within Regulatory Elements in B Cells from Primary Sjögren's Syndrome Patients.

    Science.gov (United States)

    Konsta, Orsia D; Le Dantec, Christelle; Charras, Amandine; Brooks, Wesley H; Arleevskaya, Marina I; Bordron, Anne; Renaudineau, Yves

    2015-01-01

    Recent advances in genetics have highlighted several regions and candidate genes associated with primary Sjögren's syndrome (SS), a systemic autoimmune epithelitis that combines exocrine gland dysfunctions, and focal lymphocytic infiltrations. In addition to genetic factors, it is now clear that epigenetic deregulations are present during SS and restricted to specific cell type subsets, such as lymphocytes and salivary gland epithelial cells. In this study, 72 single nucleotide polymorphisms (SNPs) associated with 43 SS gene risk factors were selected from publicly available and peer reviewed literature for further in silico analysis. SS risk variant location was tested revealing a broad distribution in coding sequences (5.6%), intronic sequences (55.6%), upstream/downstream genic regions (30.5%), and intergenic regions (8.3%). Moreover, a significant enrichment of regulatory motifs (promoter, enhancer, insulator, DNAse peak, and expression quantitative trait loci) characterizes SS risk variants (94.4%). Next, screening SNPs in high linkage disequilibrium (r (2) ≥ 0.8 in Caucasians) revealed 645 new variants including 5 SNPs with missense mutations, and indicated an enrichment of transcriptionally active motifs according to the cell type (B cells > monocytes > T cells ≫ A549). Finally, we looked at SS risk variants for histone markers in B cells (GM12878), monocytes (CD14(+)) and epithelial cells (A548). Active histone markers were associated with SS risk variants at both promoters and enhancers in B cells, and within enhancers in monocytes. In conclusion and based on the obtained in silico results that need further confirmation, associations were observed between SS genetic risk factors and epigenetic factors and these associations predominate in B cells, such as those observed at the FAM167A-BLK locus. PMID:26379672

  18. New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology

    Directory of Open Access Journals (Sweden)

    Meile Leo

    2010-07-01

    Full Text Available Abstract Background Oxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum NCC2705 were immobilized in gellan-xanthan gum gel beads and used to continuously ferment MRS medium containing increasing concentration of H2O2 from 0 to 130 ppm. Results At the beginning of the culture, high cell density of 1013 CFU per litre of reactor was tested. The continuous culture gradually adapted to increasing H2O2 concentrations. However, after increasing the H2O2 concentration to 130 ppm the OD of the culture decreased to 0. Full wash out was prevented by the immobilization of the cells in gel matrix. Hence after stopping the stress, it was possible to re-grow the cells that survived the highest lethal dose of H2O2 and to select two adapted colonies (HPR1 and HPR2 after plating of the culture effluent. In contrast to HPR1, HPR2 showed stable characteristics over at least 70 generations and exhibited also higher tolerance to O2 than non adapted wild type cells. Preliminary characterization of HPR2 was carried out by global genome expression profile analysis. Two genes coding for a protein with unknown function and possessing trans-membrane domains and an ABC-type transporter protein were overexpressed in HPR2 cells compared to wild type cells. Conclusions Our study showed that continuous culture with cell immobilization is a valid approach for selecting cells adapted to hydrogen peroxide. Elucidation of H2O2 adaptation mechanisms in HPR2 could be helpful to develop oxygen resistant bifidobacteria.

  19. Microsecond molecular dynamics simulations of the open state structure of a bacterial voltage-gated sodium channel reveal mechanisms of ion selectivity and conduction

    OpenAIRE

    Ulmschneider, Martin B.; Bagneris, Claire; McCusker, Emily C.; Ulmschneider, J.P.; Wallace, Bonnie A.

    2013-01-01

    Microsecond atomic detail equilibrium molecular dynamics simulations based on the open-state crystal structure (McCusker et al, 2012, Nature Comm) of a bacterial voltage-gated sodium channel (NavMs) have been employed to characterize the mechanisms underlying ion selectivity and conductance of the channel embedded in a lipid bilayer membrane. This approach captured the full plethora of conduction events, revealing a complex mixture of single and multi-ion phenomena, with decoupled rapid bi-di...

  20. Co-occurrence of resistance genes to antibiotics, biocides and metals reveals novel insights into their co-selection potential

    OpenAIRE

    Pal, Chandan; Bengtsson-Palme, Johan; Kristiansson, Erik; Larsson, D. G. Joakim

    2015-01-01

    Background Antibacterial biocides and metals can co-select for antibiotic resistance when bacteria harbour resistance or tolerance genes towards both types of compounds. Despite numerous case studies, systematic and quantitative data on co-occurrence of such genes on plasmids and chromosomes is lacking, as is knowledge on environments and bacterial taxa that tend to carry resistance genes to such compounds. This effectively prevents identification of risk scenarios. Therefore, we aimed to ide...

  1. ARTIFICIAL SELECTION ON RELATIVE BRAIN SIZE REVEALS A POSITIVE GENETIC CORRELATION BETWEEN BRAIN SIZE AND PROACTIVE PERSONALITY IN THE GUPPY

    OpenAIRE

    Kotrschal, Alexander; Lievens, Eva J.P.; Dahlbom, Josefin; Bundsen, Andreas; Semenova, Svetlana; Sundvik, Maria; Maklakov, Alexei A.; Winberg, Svante; Panula, Pertti; Kolm, Niclas

    2014-01-01

    Animal personalities range from individuals that are shy, cautious, and easily stressed (a "reactive" personality type) to individuals that are bold, innovative, and quick to learn novel tasks, but also prone to routine formation (a "proactive" personality type). Although personality differences should have important consequences for fitness, their underlying mechanisms remain poorly understood. Here, we investigated how genetic variation in brain size affects personality. We put selection li...

  2. Transcriptome sequencing of Eucalyptus camaldulensis seedlings subjected to water stress reveals functional single nucleotide polymorphisms and genes under selection

    OpenAIRE

    Thumma Bala R; Sharma Navin; Southerton Simon G

    2012-01-01

    Abstract Background Water stress limits plant survival and production in many parts of the world. Identification of genes and alleles responding to water stress conditions is important in breeding plants better adapted to drought. Currently there are no studies examining the transcriptome wide gene and allelic expression patterns under water stress conditions. We used RNA sequencing (RNA-seq) to identify the candidate genes and alleles and to explore the evolutionary signatures of selection. ...

  3. Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

    OpenAIRE

    Clerc, Jérôme; Groll, Michael; Illich, Damir J.; Bachmann, André S.; Huber, Robert; Schellenberg, Barbara; Dudler, Robert; Kaiser, Markus

    2009-01-01

    Syrbactins, a family of natural products belonging either to the syringolin or glidobactin class, are highly potent proteasome inhibitors. Although sharing similar structural features, they differ in their macrocyclic lactam core structure and exocyclic side chain. These structural variations critically influence inhibitory potency and proteasome subsite selectivity. Here, we describe the total synthesis of syringolin A and B, which together with enzyme kinetic and structural studies, allowed...

  4. Paired neuron recordings in the prefrontal and inferotemporal cortices reveal that spatial selection precedes object identification during visual search

    OpenAIRE

    Monosov, Ilya E.; David L Sheinberg; Thompson, Kirk G.

    2010-01-01

    We addressed the question of how we locate and identify objects in complex natural environments by simultaneously recording single neurons from two brain regions that play different roles in this familiar activity—the frontal eye field (FEF), an area in the prefrontal cortex that is involved in visual spatial selection, and the inferotemporal cortex (IT), which is involved in object recognition—in monkeys performing a covert visual search task. Although the monkeys reported object identity, n...

  5. Selection for cuticular melanism reveals immune function and life-history trade-offs in Spodoptera littoralis

    OpenAIRE

    Cotter, S. C.; Myatt, J. P.; Benskin, C. M. H.; Wilson, K.

    2008-01-01

    Several insect species show an increase in cuticular melanism in response to high densities. In some species, there is evidence that this melanism is correlated with an up-regulation of certain immune system components, particularly phenoloxidase (PO) activity, and with the down-regulation of lysozyme activity, suggesting a trade-off between the two traits. As melanism has a genetic component, we selected both melanic and nonmelanic lines of the phase-polyphenic lepidopteran, Spodoptera litto...

  6. A Novel Binding Mode Reveals Two Distinct Classes of NMDA Receptor GluN2B-selective Antagonists.

    Science.gov (United States)

    Stroebel, David; Buhl, Derek L; Knafels, John D; Chanda, Pranab K; Green, Michael; Sciabola, Simone; Mony, Laetitia; Paoletti, Pierre; Pandit, Jayvardhan

    2016-05-01

    N-methyl-d-aspartate receptors (NMDARs) are glutamate-gated ion channels that play key roles in brain physiology and pathology. Because numerous pathologic conditions involve NMDAR overactivation, subunit-selective antagonists hold strong therapeutic potential, although clinical successes remain limited. Among the most promising NMDAR-targeting drugs are allosteric inhibitors of GluN2B-containing receptors. Since the discovery of ifenprodil, a range of GluN2B-selective compounds with strikingly different structural motifs have been identified. This molecular diversity raises the possibility of distinct binding sites, although supporting data are lacking. Using X-ray crystallography, we show that EVT-101, a GluN2B antagonist structurally unrelated to the classic phenylethanolamine pharmacophore, binds at the same GluN1/GluN2B dimer interface as ifenprodil but adopts a remarkably different binding mode involving a distinct subcavity and receptor interactions. Mutagenesis experiments demonstrate that this novel binding site is physiologically relevant. Moreover, in silico docking unveils that GluN2B-selective antagonists broadly divide into two distinct classes according to binding pose. These data widen the allosteric and pharmacological landscape of NMDARs and offer a renewed structural framework for designing next-generation GluN2B antagonists with therapeutic value for brain disorders. PMID:26912815

  7. Discovery of the surface polarity gradient on iridescent Morpho butterfly scales reveals a mechanism of their selective vapor response.

    Science.gov (United States)

    Potyrailo, Radislav A; Starkey, Timothy A; Vukusic, Peter; Ghiradella, Helen; Vasudev, Milana; Bunning, Timothy; Naik, Rajesh R; Tang, Zhexiong; Larsen, Michael; Deng, Tao; Zhong, Sheng; Palacios, Manuel; Grande, James C; Zorn, Gilad; Goddard, Gregory; Zalubovsky, Sergey

    2013-09-24

    For almost a century, the iridescence of tropical Morpho butterfly scales has been known to originate from 3D vertical ridge structures of stacked periodic layers of cuticle separated by air gaps. Here we describe a biological pattern of surface functionality that we have found in these photonic structures. This pattern is a gradient of surface polarity of the ridge structures that runs from their polar tops to their less-polar bottoms. This finding shows a biological pattern design that could stimulate numerous technological applications ranging from photonic security tags to self-cleaning surfaces, gas separators, protective clothing, sensors, and many others. As an important first step, this biomaterial property and our knowledge of its basis has allowed us to unveil a general mechanism of selective vapor response observed in the photonic Morpho nanostructures. This mechanism of selective vapor response brings a multivariable perspective for sensing, where selectivity is achieved within a single chemically graded nanostructured sensing unit, rather than from an array of separate sensors. PMID:24019497

  8. High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells.

    Directory of Open Access Journals (Sweden)

    Yo Saito

    Full Text Available Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with GATA-2 intronic enhancer (+9.9 kb/1S; therefore, we established a cell line capable of stably expressing +9.9 kb/1S-Luciferase. Subsequently, we screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells. These results provide novel insights into and further identify the regulatory mechanism of GATA-2.

  9. Relative contribution of "determinant selection" and "holes in the T-cell repertoire" to T-cell responses

    DEFF Research Database (Denmark)

    Schaeffer, E B; Sette, A; Johnson, D L; Bekoff, M C; Smith, J A; Grey, H M; Buus, S

    1989-01-01

    for a large universe of antigens. On the other hand, since the Ia molecules cannot distinguish between self and non-self, not all antigen-Ia interactions would be permitted to elicit a T-cell response. It appears that both Ia binding ("determinant selection") and T-cell repertoire act in concert to...

  10. Energy deposition in selected-mammalian cell for several-MeV single-proton beam

    Science.gov (United States)

    Ding, K.; Yu, Z.

    2007-05-01

    The phenomena resulting from interaction between ion beam and mammalian cell pose important problems for biological applications. Classic Bethe-Bloch theory utilizing attached V79 mammalian cell has been conducted in order to establish the stopping powers of the mammalian cell for several-MeV single-proton microbeam. Based on the biological structure of the mammalian cell, a physical model is proposed which presumes that the attached cell is simple MWM model. According to this model and Monte Carlo simulation, we studied the energy deposition and its ratio on the selected attached mammalian cell for MeV proton implantation.

  11. Proteotranscriptomic Analysis Reveals Stage Specific Changes in the Molecular Landscape of Clear-Cell Renal Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Benjamin A Neely

    Full Text Available Renal cell carcinoma comprises 2 to 3% of malignancies in adults with the most prevalent subtype being clear-cell RCC (ccRCC. This type of cancer is well characterized at the genomic and transcriptomic level and is associated with a loss of VHL that results in stabilization of HIF1. The current study focused on evaluating ccRCC stage dependent changes at the proteome level to provide insight into the molecular pathogenesis of ccRCC progression. To accomplish this, label-free proteomics was used to characterize matched tumor and normal-adjacent tissues from 84 patients with stage I to IV ccRCC. Using pooled samples 1551 proteins were identified, of which 290 were differentially abundant, while 783 proteins were identified using individual samples, with 344 being differentially abundant. These 344 differentially abundant proteins were enriched in metabolic pathways and further examination revealed metabolic dysfunction consistent with the Warburg effect. Additionally, the protein data indicated activation of ESRRA and ESRRG, and HIF1A, as well as inhibition of FOXA1, MAPK1 and WISP2. A subset analysis of complementary gene expression array data on 47 pairs of these same tissues indicated similar upstream changes, such as increased HIF1A activation with stage, though ESRRA and ESRRG activation and FOXA1 inhibition were not predicted from the transcriptomic data. The activation of ESRRA and ESRRG implied that HIF2A may also be activated during later stages of ccRCC, which was confirmed in the transcriptional analysis. This combined analysis highlights the importance of HIF1A and HIF2A in developing the ccRCC molecular phenotype as well as the potential involvement of ESRRA and ESRRG in driving these changes. In addition, cofilin-1, profilin-1, nicotinamide N-methyltransferase, and fructose-bisphosphate aldolase A were identified as candidate markers of late stage ccRCC. Utilization of data collected from heterogeneous biological domains strengthened

  12. Proteotranscriptomic Analysis Reveals Stage Specific Changes in the Molecular Landscape of Clear-Cell Renal Cell Carcinoma

    Science.gov (United States)

    Wilkins, Christopher E.; Marlow, Laura A.; Malyarenko, Dariya; Kim, Yunee; Ignatchenko, Alexandr; Sasinowska, Heather; Sasinowski, Maciek; Nyalwidhe, Julius O.; Kislinger, Thomas; Copland, John A.; Drake, Richard R.

    2016-01-01

    Renal cell carcinoma comprises 2 to 3% of malignancies in adults with the most prevalent subtype being clear-cell RCC (ccRCC). This type of cancer is well characterized at the genomic and transcriptomic level and is associated with a loss of VHL that results in stabilization of HIF1. The current study focused on evaluating ccRCC stage dependent changes at the proteome level to provide insight into the molecular pathogenesis of ccRCC progression. To accomplish this, label-free proteomics was used to characterize matched tumor and normal-adjacent tissues from 84 patients with stage I to IV ccRCC. Using pooled samples 1551 proteins were identified, of which 290 were differentially abundant, while 783 proteins were identified using individual samples, with 344 being differentially abundant. These 344 differentially abundant proteins were enriched in metabolic pathways and further examination revealed metabolic dysfunction consistent with the Warburg effect. Additionally, the protein data indicated activation of ESRRA and ESRRG, and HIF1A, as well as inhibition of FOXA1, MAPK1 and WISP2. A subset analysis of complementary gene expression array data on 47 pairs of these same tissues indicated similar upstream changes, such as increased HIF1A activation with stage, though ESRRA and ESRRG activation and FOXA1 inhibition were not predicted from the transcriptomic data. The activation of ESRRA and ESRRG implied that HIF2A may also be activated during later stages of ccRCC, which was confirmed in the transcriptional analysis. This combined analysis highlights the importance of HIF1A and HIF2A in developing the ccRCC molecular phenotype as well as the potential involvement of ESRRA and ESRRG in driving these changes. In addition, cofilin-1, profilin-1, nicotinamide N-methyltransferase, and fructose-bisphosphate aldolase A were identified as candidate markers of late stage ccRCC. Utilization of data collected from heterogeneous biological domains strengthened the findings from

  13. Selective Cancer Targeting via Aberrant Behavior of Cancer Cell-associated Glucocorticoid Receptor

    OpenAIRE

    Mukherjee, Amarnath; Narayan, Kumar P; Pal, Krishnendu; Kumar, Jerald M.; Rangaraj, Nandini; Shasi V Kalivendi; Banerjee, Rajkumar

    2009-01-01

    Glucocorticoid receptors (GRs) are ubiquitous, nuclear hormone receptors residing in cell types of both cancer and noncancerous origin. It is not known whether cancer cell–associated GR alone can be selectively manipulated for delivery of exogenous genes to its nucleus for eliciting anticancer effect. We find that GR ligand, dexamethasone (Dex) in association with cationic lipoplex (termed as targeted lipoplex) could selectively manipulate GR in cancer cells alone for the delivery of transgen...

  14. A mammary repopulating cell population characterized in mammary anlagen reveals essential mammary stroma for morphogenesis.

    Science.gov (United States)

    Song, Jiazhe; Xue, Kai; She, Ji; Ding, Fangrong; Li, Song; Shangguan, Rulan; Dai, Yunping; Du, Liying; Li, Ning

    2014-09-10

    The cells with mammary repopulating capability can achieve mammary gland morphogenesis in a suitable cellular microenvironment. Using cell surface markers of CD24, CD29 and CD49f, mouse mammary repopulating unit (MRU) has been identified in adult mammary epithelium and late embryonic mammary bud epithelium. However, embryonic MRU remains to be fully characterized at earlier mammary anlagen stage. Here we isolated discrete populations of E14.5 mouse mammary anlagen cells. Only Lin(-)CD24(med)CD29(+) cell population was predicted as E14.5 MRU by examining their capacities of forming mammosphere and repopulating cleared mammary fat pad in vivo. However, when we characterized gene expressions of this E14.5 cell population by comparing with adult mouse MRU (Lin(-)CD24(+)CD29(hi)), the gene profiling of these two cell populations exhibited great differences. Real-time PCR and immunostaining assays uncovered that E14.5 Lin(-)CD24(med)CD29(+) cell population was a heterogeneous stroma-enriched cell population. Then, limiting dilutions and single-cell assays also confirmed that E14.5 Lin(-)CD24(med)CD29(+) cell population possessed low proportion of stem cells. In summary, heterogeneous Lin(-)CD24(med)CD29(+) cell population exhibited mammary repopulating ability in E14.5 mammary anlagen, implying that only suitable mammary stroma could enable mammary gland morphogenesis, which relied on the interaction between rare stem cells and microenvironment. PMID:24954407

  15. Selective expression of mutated p53 in human cells immortalized with either 4-nitroquinoline 1-oxide or 60Co gamma rays

    International Nuclear Information System (INIS)

    Many studies on in vitro transformation of human cells indicate that the cells must be immortalized before they can be neoplastically transformed, indicating that immortalization is a critical step in multistep neoplastic transformation of human cells. We immortalized three human cell lines by repeated treatment with either 60Co gamma rays or a chemical carcinogen, 4-nitroquinoline 1-oxide, and found that all three immortalized cell lines have mutations in the tumor suppressor gene, p53. Direct sequencing of the reverse-transcribed mRNA and immunoprecipitation of p53 protein revealed that mutant p53 is selectively expressed in all the immortalized cell lines, whereas the genomic fragments of the immortalized cells contain wild-type and mutated p53 alleles. Although the mutated p53 is selectively expressed in the immortalized cells, expression of the wild-type p53 was induced by treatment of the cells with a hypomethylating reagent, 5-azacytidine, indicating that the wild-type p53 allele might be inactivated by hypermethylation of DNA. Actually, the entire genomic locus including the promoter region of p53 is hypermethylated in all the immortalized cell lines. Expression and phosphorylation of Rb was normal in these three cell lines. Thus, inactivation of both wild type p53 alleles and selective expression of mutated p53 seem to be key factors in the immortalization of human fibroblasts. (author)

  16. Double suicide genes selectively kill human umbilical vein endothelial cells

    OpenAIRE

    Liu Lunxu; Wang Yanping; Mei Longyong; Jia Weiguo; Che Guowei

    2011-01-01

    Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli) cytosine deaminase (CD) gene and the herpes simplex virus-thymidine kinase (TK) gene were cloned using polymerase chain reaction (PCR). Plasmid pKDR-CDglyTK was constructed wi...

  17. Combinational Effect of Cell Adhesion Biomolecules and Their Immobilized Polymer Property to Enhance Cell-Selective Adhesion

    Directory of Open Access Journals (Sweden)

    Rio Kurimoto

    2016-01-01

    Full Text Available Although surface immobilization of medical devices with bioactive molecules is one of the most widely used strategies to improve biocompatibility, the physicochemical properties of the biomaterials significantly impact the activity of the immobilized molecules. Herein we investigate the combinational effects of cell-selective biomolecules and the hydrophobicity/hydrophilicity of the polymeric substrate on selective adhesion of endothelial cells (ECs, fibroblasts (FBs, and smooth muscle cells (SMCs. To control the polymeric substrate, biomolecules are immobilized on thermoresponsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide (poly(NIPAAm-co-CIPAAm-grafted glass surfaces. By switching the molecular conformation of the biomolecule-immobilized polymers, the cell-selective adhesion performances are evaluated. In case of RGDS (Arg-Gly-Asp-Ser peptide-immobilized surfaces, all cell types adhere well regardless of the surface hydrophobicity. On the other hand, a tri-Arg-immobilized surface exhibits FB-selectivity when the surface is hydrophilic. Additionally, a tri-Ile-immobilized surface exhibits EC-selective cell adhesion when the surface is hydrophobic. We believe that the proposed concept, which is used to investigate the biomolecule-immobilized surface combination, is important to produce new biomaterials, which are highly demanded for medical implants and tissue engineering.

  18. Enhanced transfection of cell lines from Atlantic salmon through nucoleofection and antibiotic selection

    Directory of Open Access Journals (Sweden)

    Mjaaland Siri

    2011-05-01

    Full Text Available Abstract Background Cell lines from Atlantic salmon kidney have made it possible to culture and study infectious salmon anemia virus (ISAV, an aquatic orthomyxovirus affecting farmed Atlantic salmon. However, transfection of these cells using calcium phosphate precipitation or lipid-based reagents shows very low transfection efficiency. The Amaxa Nucleofector technology™ is an electroporation technique that has been shown to be efficient for gene transfer into primary cells and hard to transfect cell lines. Findings Here we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry. Depending on the plasmid promoter, TO cells could be transfected transiently with an efficiency ranging from 11.6% to 90.8% with good viability, using Amaxa's cell line nucleofector solution T and program T-20. A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells. Conclusions The results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin.

  19. Impact of selection for decreased somatic cell score on productive life and culling for mastitis

    Science.gov (United States)

    Impact of continued selection for decreased somatic cell score (SCS) was examined to determine if such selection resulted in greater mastitis susceptibility and shorter productive life (PL). Holstein artificial-insemination bulls with a predicted transmitting ability (PTA) for SCS based on >=35 daug...

  20. A comparative proteomic analysis of the simple amino acid repeat distributions in Plasmodia reveals lineage specific amino acid selection.

    Directory of Open Access Journals (Sweden)

    Andrew R Dalby

    Full Text Available BACKGROUND: Microsatellites have been used extensively in the field of comparative genomics. By studying microsatellites in coding regions we have a simple model of how genotypic changes undergo selection as they are directly expressed in the phenotype as altered proteins. The simplest of these tandem repeats in coding regions are the tri-nucleotide repeats which produce a repeat of a single amino acid when translated into proteins. Tri-nucleotide repeats are often disease associated, and are also known to be unstable to both expansion and contraction. This makes them sensitive markers for studying proteome evolution, in closely related species. RESULTS: The evolutionary history of the family of malarial causing parasites Plasmodia is complex because of the life-cycle of the organism, where it interacts with a number of different hosts and goes through a series of tissue specific stages. This study shows that the divergence between the primate and rodent malarial parasites has resulted in a lineage specific change in the simple amino acid repeat distribution that is correlated to A-T content. The paper also shows that this altered use of amino acids in SAARs is consistent with the repeat distributions being under selective pressure. CONCLUSIONS: The study shows that simple amino acid repeat distributions can be used to group related species and to examine their phylogenetic relationships. This study also shows that an outgroup species with a similar A-T content can be distinguished based only on the amino acid usage in repeats, and suggest that this might be a useful feature for proteome clustering. The lineage specific use of amino acids in repeat regions suggests that comparative studies of SAAR distributions between proteomes gives an insight into the mechanisms of expansion and the selective pressures acting on the organism.

  1. A synthetic interaction screen identifies factors selectively required for proliferation and TERT transcription in p53-deficient human cancer cells.

    Directory of Open Access Journals (Sweden)

    Li Xie

    Full Text Available Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi-based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53- human cancer cells. We find that compared to p53-competent (p53+ human cancer cell lines, diverse p53- human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53- cells, RNAi-mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53- but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53- cancer cells.

  2. agged tumor cells reveal regulatory steps during earliest stages of tumor progression and micrometastasis

    OpenAIRE

    Culp, L A; Lin, W.C.

    1999-01-01

    Histochemical marker genes were used to "tag" mouse fibrosarcoma or human neuroblastoma cells, providing a better understanding of their subsequent progression and metastasis mechanisms in nude mice. Micrometastases in the lung were initiated from clusters of 2-6 cells rather than single cells in most cases; tumor cells were also visualized binding to the endothelium of small blood vessels to initiate these micrometastases. Shortterm, these mechanisms relied ...

  3. Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry

    OpenAIRE

    Horowitz, Amir; Strauss-Albee, Dara M.; Leipold, Michael; Kubo, Jessica; Nemat-Gorgani, Neda; DOGAN, Ozge C.; Dekker, Cornelia L; Mackey, Sally; Maecker, Holden; Swan, Gary E.; Davis, Mark M.; Norman, Paul J.; Guethlein, Lisbeth A.; Desai, Manisha; Parham, Peter

    2013-01-01

    Natural Killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 35 parameters, including 28 NK cell receptors, on perip...

  4. Selective MS screening reveals a sex pheromone in Caenorhabditis briggsae and species-specificity in indole ascaroside signalling.

    Science.gov (United States)

    Dong, Chuanfu; Dolke, Franziska; von Reuss, Stephan H

    2016-08-14

    The indole ascarosides (icas) represent a highly potent class of nematode-derived modular signalling components that integrate structural inputs from amino acid, carbohydrate, and fatty acid metabolism. Comparative analysis of the crude exo-metabolome of hermaphroditic Caenorhabditis briggsae using a highly sensitive mass spectrometric screen reveals an indole ascaroside blend dominated by two new components. The structures of isolated icas#2 and icas#6.2 were determined by NMR spectroscopy and confirmed by total synthesis and chemical correlation. Low atto- to femtomolar amounts of icas#2 and icas#6.2 act in synergism to attract males indicating a function as sex pheromone. Comparative analysis of 14 Caenorhabditis species further demonstrates that species-specific indole ascaroside biosynthesis is highly conserved in the Elegans group. Functional characterization of the dominating indole ascarosides icas#2, icas#3, and icas#9 reveals a high degree of species-specificity and considerable variability with respect to gender-specificity, thus, confirming that indole ascarosides modulate different biological functions within the Elegans group. Although the nematode response was usually most pronounced towards conspecific signals, Caenorhabditis brenneri, the only species of the Elegans group that does not produce any indole ascarosides, exhibits a robust response to icas#2 suggesting the potential for interspecies interactions. PMID:27381649

  5. Computational models reveal a passive mechanism for cell migration in the crypt.

    Directory of Open Access Journals (Sweden)

    Sara-Jane Dunn

    Full Text Available Cell migration in the intestinal crypt is essential for the regular renewal of the epithelium, and the continued upward movement of cells is a key characteristic of healthy crypt dynamics. However, the driving force behind this migration is unknown. Possibilities include mitotic pressure, active movement driven by motility cues, or negative pressure arising from cell loss at the crypt collar. It is possible that a combination of factors together coordinate migration. Here, three different computational models are used to provide insight into the mechanisms that underpin cell movement in the crypt, by examining the consequence of eliminating cell division on cell movement. Computational simulations agree with existing experimental results, confirming that migration can continue in the absence of mitosis. Importantly, however, simulations allow us to infer mechanisms that are sufficient to generate cell movement, which is not possible through experimental observation alone. The results produced by the three models agree and suggest that cell loss due to apoptosis and extrusion at the crypt collar relieves cell compression below, allowing cells to expand and move upwards. This finding suggests that future experiments should focus on the role of apoptosis and cell extrusion in controlling cell migration in the crypt.

  6. The volumes and transcript counts of single cells reveal concentration homeostasis and capture biological noise

    NARCIS (Netherlands)

    H. Kempe; A. Schwabe; F. Crémazy; P.J. Verschure; F.J. Bruggeman

    2015-01-01

    Transcriptional stochasticity can be measured by counting the number of mRNA molecules per cell. Cell-to-cell variability is best captured in terms of concentration rather than molecule counts, because reaction rates depend on concentrations. We combined single-molecule mRNA counting with single-cel

  7. Selective apoptosis induction in MCF-7 cell line by truncated minimal functional region of Apoptin

    International Nuclear Information System (INIS)

    Chicken Anemia Virus (CAV) VP3 protein (also known as Apoptin), a basic and proline-rich protein has a unique capability in inducing apoptosis in cancer cells but not in normal cells. Five truncated Apoptin proteins were analyzed to determine their selective ability to migrate into the nucleus of human breast adenocarcinoma MCF-7 cells for inducing apoptosis. For identification of the minimal selective domain for apoptosis, the wild-type Apoptin gene had been reconstructed by PCR to generate segmental deletions at the N’ terminal and linked with nuclear localization sites (NLS1 and NLS2). All the constructs were fused with maltose-binding protein gene and individually expressed by in vitro Rapid Translation System. Standardized dose of proteins were delivered into human breast adenocarcinoma MCF-7 cells and control human liver Chang cells by cytoplasmic microinjection, and subsequently observed for selective apoptosis effect. Three of the truncated Apoptin proteins with N-terminal deletions spanning amino acid 32–83 retained the cancer selective nature of wild-type Apoptin. The proteins were successfully translocated to the nucleus of MCF-7 cells initiating apoptosis, whereas non-toxic cytoplasmic retention was observed in normal Chang cells. Whilst these truncated proteins retained the tumour-specific death effector ability, the specificity for MCF-7 cells was lost in two other truncated proteins that harbor deletions at amino acid 1–31. The detection of apoptosing normal Chang cells and MCF-7 cells upon cytoplasmic microinjection of these proteins implicated a loss in Apoptin’s signature targeting activity. Therefore, the critical stretch spanning amino acid 1–31 at the upstream of a known hydrophobic leucine-rich stretch (LRS) was strongly suggested as one of the prerequisite region in Apoptin for cancer targeting. Identification of this selective domain provides a platform for developing small targets to facilitating carrier-mediated-transport across

  8. Landscape genomics and biased FST approaches reveal single nucleotide polymorphisms under selection in goat breeds of North-East Mediterranean

    Directory of Open Access Journals (Sweden)

    Joost Stephane

    2009-02-01

    Full Text Available Abstract Background In this study we compare outlier loci detected using a FST based method with those identified by a recently described method based on spatial analysis (SAM. We tested a panel of single nucleotide polymorphisms (SNPs previously genotyped in individuals of goat breeds of southern areas of the Mediterranean basin (Italy, Greece and Albania. We evaluate how the SAM method performs with SNPs, which are increasingly employed due to their high number, low cost and easy of scoring. Results The combined use of the two outlier detection approaches, never tested before using SNP polymorphisms, resulted in the identification of the same three loci involved in milk and meat quality data by using the two methods, while the FST based method identified 3 more loci as under selection sweep in the breeds examined. Conclusion Data appear congruent by using the two methods for FST values exceeding the 99% confidence limits. The methods of FST and SAM can independently detect signatures of selection and therefore can reduce the probability of finding false positives if employed together. The outlier loci identified in this study could indicate adaptive variation in the analysed species, characterized by a large range of climatic conditions in the rearing areas and by a history of intense trade, that implies plasticity in adapting to new environments.

  9. A novel molecule integrating therapeutic and diagnostic activities reveals multiple aspects of stem cell-based therapy.

    Science.gov (United States)

    Hingtgen, Shawn D; Kasmieh, Randa; van de Water, Jeroen; Weissleder, Ralph; Shah, Khalid

    2010-04-01

    Stem cells are promising therapeutic delivery vehicles; however pre-clinical and clinical applications of stem cell-based therapy would benefit significantly from the ability to simultaneously determine therapeutic efficacy and pharmacokinetics of therapies delivered by engineered stem cells. In this study, we engineered and screened numerous fusion variants that contained therapeutic (TRAIL) and diagnostic (luciferase) domains designed to allow simultaneous investigation of multiple events in stem cell-based therapy in vivo. When various stem cell lines were engineered with the optimized molecule, SRL(O)L(2)TR, diagnostic imaging showed marked differences in the levels and duration of secretion between stem cell lines, while the therapeutic activity of the molecule showed the different secretion levels translated to significant variability in tumor cell killing. In vivo, simultaneous diagnostic and therapeutic monitoring revealed that stem cell-based delivery significantly improved pharmacokinetics and anti-tumor effectiveness of the therapy compared to intravenous or intratumoral delivery. As treatment for highly malignant brain tumor xenografts, tracking SRL(O)L(2)TR showed stable stem cell-mediated delivery significantly regressed peripheral and intracranial tumors. Together, the integrated diagnostic and therapeutic properties of SRL(O)L(2)TR answer critical questions necessary for successful utilization of stem cells as novel therapeutic vehicles. PMID:20127797

  10. Lineage relationship of CD8+ T cell subsets is revealed by progressive changes in the epigenetic landscape

    Science.gov (United States)

    Crompton, Joseph G.; Narayanan, Manikandan; Cuddapah, Suresh; Roychoudhuri, Rahul; Ji, Yun; Yang, Wenjing; Patel, Shashank J.; Sukumar, Madhusudhanan; Palmer, Douglas C.; Peng, Weiqun; Wang, Ena; Marincola, Francesco M.; Klebanoff, Christopher A.; Zhao, Keji; Tsang, John S.; Gattinoni, Luca; Restifo, Nicholas P.

    2016-01-01

    To better elucidate epigenetic mechanisms that correlate with the dynamic gene expression program observed upon T-cell differentiation, we investigated the genomic landscape of histone modifications in naive and memory CD8+ T cells. Using a ChIP-Seq approach coupled with global gene expression profiling, we generated genome-wide histone H3 lysine 4 (H3K4me3) and H3 lysine 27 (H3K27me3) trimethylation maps in naive, T memory stem cells, central memory cells, and effector memory cells in order to gain insight into how histone architecture is remodeled during T cell differentiation. We show that H3K4me3 histone modifications are associated with activation of genes, while H3K27me3 is negatively correlated with gene expression at canonical loci and enhancers associated with T-cell metabolism, effector function, and memory. Our results also reveal histone modifications and gene expression signatures that distinguish the recently identified T memory stem cells from other CD8+ T-cell subsets. Taken together, our results suggest that CD8+ lymphocytes undergo chromatin remodeling in a progressive fashion. These findings have major implications for our understanding of peripheral T-cell ontogeny and the formation of immunological memory. PMID:25914936

  11. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    International Nuclear Information System (INIS)

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus

  12. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    Energy Technology Data Exchange (ETDEWEB)

    Horita, Nobukatsu [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Hayashi, Ryohei [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Department of Gastroenterology and Metabolism, Hiroshima University (Japan); Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Okamoto, Ryuichi; Nakamura, Tetsuya [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan)

    2014-11-28

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

  13. Cytokine-dependent and–independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi-infected host cells by comparative mRNA profiling

    Directory of Open Access Journals (Sweden)

    Burleigh Barbara A

    2009-05-01

    Full Text Available Abstract Background The requirements for growth and survival of the intracellular pathogen Trypanosoma cruzi within mammalian host cells are poorly understood. Transcriptional profiling of the host cell response to infection serves as a rapid read-out for perturbation of host physiology that, in part, reflects adaptation to the infective process. Using Affymetrix oligonucleotide array analysis we identified common and disparate host cell responses triggered by T. cruzi infection of phenotypically diverse human cell types. Results We report significant changes in transcript abundance in T. cruzi-infected fibroblasts, endothelial cells and smooth muscle cells (2852, 2155 and 531 genes respectively; fold-change ≥ 2, p-value T. cruzi-infected fibroblasts and endothelial cells transwell plates were used to distinguish cytokine-dependent and -independent gene expression profiles. This approach revealed the induction of metabolic and signaling pathways involved in cell proliferation, amino acid catabolism and response to wounding as common themes in T. cruzi-infected cells. In addition, the downregulation of genes involved in mitotic cell cycle and cell division predicted that T. cruzi infection may impede host cell cycle progression. The observation of impaired cytokinesis in T. cruzi-infected cells, following nuclear replication, confirmed this prediction. Conclusion Metabolic pathways and cellular processes were identified as significantly altered at the transcriptional level in response to T. cruzi infection in a cytokine-independent manner. Several of these alterations are supported by previous studies of T. cruzi metabolic requirements or effects on the host. However, our methods also revealed a T. cruzi-dependent block in the host cell cycle, at the level of cytokinesis, previously unrecognized for this pathogen-host cell interaction.

  14. Expression weighted cell type enrichments reveal genetic and cellular nature of major brain disorders

    Directory of Open Access Journals (Sweden)

    Nathan Gerald Skene

    2016-01-01

    Full Text Available The cell types that trigger the primary pathology in many brain diseases remain largely unknown. One route to understanding the primary pathological cell type for a particular disease is to identify the cells expressing susceptibility genes. Although this is straightforward for monogenic conditions where the causative mutation may alter expression of a cell type specific marker, methods are required for the common polygenic disorders. We developed the Expression Weighted Cell Type Enrichment (EWCE method that uses single cell transcriptomes to generate the probability distribution associated with a gene list having an average level of expression within a cell type. Following validation, we applied EWCE to human genetic data from cases of epilepsy, Schizophrenia, Autism, Intellectual Disability, Alzheimer’s disease, Multiple Sclerosis and anxiety disorders. Genetic susceptibility primarily affected microglia in Alzheimer’s and Multiple Sclerosis; was shared between interneurons and pyramidal neurons in Autism and Schizophrenia; while intellectual disabilities and epilepsy were attributable to a range of cell-types, with the strongest enrichment in interneurons. We hypothesised that the primary cell type pathology could trigger secondary changes in other cell types and these could be detected by applying EWCE to transcriptome data from diseased tissue. In Autism, Schizophrenia and Alzheimer’s disease we find evidence of pathological changes in all of the major brain cell types. These findings give novel insight into the cellular origins and progression in common brain disorders. The methods can be applied to any tissue and disorder and have applications in validating mouse models.

  15. Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease

    Science.gov (United States)

    Li, Wei; Liu, Liangyi; Gomez, Aurelie; Zhang, Jilu; Ramadan, Abdulraouf; Zhang, Qing; Choi, Sung W.; Zhang, Peng; Greenson, Joel K.; Liu, Chen; Jiang, Di; Virts, Elizabeth; Kelich, Stephanie L.; Chu, Hong Wei; Flynn, Ryan; Blazar, Bruce R.; Hanenberg, Helmut; Hanash, Samir; Paczesny, Sophie

    2016-01-01

    Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4+CD146+CCR5+ T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4+CD146+CCR5+ T cell population toward CCL14. Mice that received CD146 shRNA–transduced human T cells did not lose weight, showed better survival, and had fewer CD4+CD146+CCR5+ T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA– transduced human T cells. Furthermore, the frequency of CD4+CD146+CCR5+ Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT. PMID:27195312

  16. A High-Dimensional Atlas of Human T Cell Diversity Reveals Tissue-Specific Trafficking and Cytokine Signatures.

    Science.gov (United States)

    Wong, Michael Thomas; Ong, David Eng Hui; Lim, Frances Sheau Huei; Teng, Karen Wei Weng; McGovern, Naomi; Narayanan, Sriram; Ho, Wen Qi; Cerny, Daniela; Tan, Henry Kun Kiaang; Anicete, Rosslyn; Tan, Bien Keem; Lim, Tony Kiat Hon; Chan, Chung Yip; Cheow, Peng Chung; Lee, Ser Yee; Takano, Angela; Tan, Eng-Huat; Tam, John Kit Chung; Tan, Ern Yu; Chan, Jerry Kok Yen; Fink, Katja; Bertoletti, Antonio; Ginhoux, Florent; Curotto de Lafaille, Maria Alicia; Newell, Evan William

    2016-08-16

    Depending on the tissue microenvironment, T cells can differentiate into highly diverse subsets expressing unique trafficking receptors and cytokines. Studies of human lymphocytes have primarily focused on a limited number of parameters in blood, representing an incomplete view of the human immune system. Here, we have utilized mass cytometry to simultaneously analyze T cell trafficking and functional markers across eight different human tissues, including blood, lymphoid, and non-lymphoid tissues. These data have revealed that combinatorial expression of trafficking receptors and cytokines better defines tissue specificity. Notably, we identified numerous T helper cell subsets with overlapping cytokine expression, but only specific cytokine combinations are secreted regardless of tissue type. This indicates that T cell lineages defined in mouse models cannot be clearly distinguished in humans. Overall, our data uncover a plethora of tissue immune signatures and provide a systemic map of how T cell phenotypes are altered throughout the human body. PMID:27521270

  17. Selective enrichment of hepatoeytes from mouse embryonic stem cells with a culture system containing cholestatic serum

    Institute of Scientific and Technical Information of China (English)

    Jun MIN; Er-wei SONG; Ji-sheng CHEN; Chang-zhen SHANG; Ya-jin CHEN; Lei ZHANG; Lu LIU; Xiao-geng DENG; Mei YANG; Dong-ping CHEN; Jun CAO

    2007-01-01

    Aim: There is increasing evidence indicating that embryonic stem (ES) cells are capable of differentiating into hepatocyte-like cells in vitro. However, it is neces- sary to improve the differentiation efficiency so as to promote the clinical application. Here, we report an efficient culture system to support hepatocyte differentiation from ES cells by utilizing cholestatic serum. Methods: One week after the induction of El4 mouse ES cells into hepatocytes with sodium butyrate, cholestatic serum was added into the culture system at various concentrations and hepatocyte-like cells were induced to proliferate. The morphological and phenotypic markers of hepatocytes were characterized using light microscopy, immunocytochemistry, and RT-PCR, respectively. The function of glycogen stor- age of the differentiated cells was detected by Periodic acid-Schiff (PAS) reaction, and the ratio of hepatic differentiation was determined by counting the albumin and PAS-positive cells. Results: In the presence of conditional selective medium containing cholestatic serum, numerous epithelial cells resembling hepatocytes were observed. The RT-PCR analysis showed that undifferentiated ES cells did not express any hepatic-specific markers; however, in the presence of sodium butyrate and conditional selective medium containing cholestatic serum, hepatic differentiation markers were detected. Immunofluorescence staining showed that those ES-derived hepatocytes were α-fetoprotein, albumin, and cytokeratin 18 positive, with the ability of storing glycogen. Further determination of the hepatic differentiation ratio showed that the application of cholestatic serum efficiently enriched ES-derived hepatocyte-like cells by inducing lineage differentiation and enhancing lineage proliferation. Conclusion: The conditional selective medium containing cholestatic serum is optimal to selectively enrich hepatocyte-like cells from mixed differentiated ES cells, which may provide a novel method to

  18. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: improving the efficiency of cell line generation.

    Science.gov (United States)

    Porter, Alison J; Racher, Andrew J; Preziosi, Richard; Dickson, Alan J

    2010-01-01

    Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. PMID:20623584

  19. Propane Fuel Cells: Selectivity for Partial or Complete Reaction

    Directory of Open Access Journals (Sweden)

    Shadi Vafaeyan

    2014-01-01

    Full Text Available The use of propane fuel in high temperature (120°C polymer electrolyte membrane (PEM fuel cells that do not require a platinum group metal catalyst is being investigated in our laboratory. Density functional theory (DFT was used to determine propane adsorption energies, desorption energies, and transition state energies for both dehydrogenation and hydroxylation reactions on a Ni(100 anode catalyst surface. The Boltzmann factor for the hydroxylation of a propyl species to form propanol and its subsequent desorption was compared to that for the dehydrogenation of a propyl species. The large ratio of the respective Boltzmann factors indicated that the formation of a completely reacted product (carbon dioxide is much more likely than the formation of partially reacted products (alcohols, aldehydes, carboxylic acids, and carbon monoxide. That finding is evidence for the major proportion of the chemical energy of the propane fuel being converted to either electrical or thermal energy in the fuel cell rather than remaining unused when partially reacted species are formed.

  20. Determination of synthetic lethal interactions in KRAS oncogene-dependent cancer cells reveals novel therapeutic targeting strategies

    Institute of Scientific and Technical Information of China (English)

    Michael Steckel; Julian Downward; David C Hancock; Miriam Molina-Arcas; Britta Weigelt; Michaela Marani; Patricia H Warne; Hanna Kuznetsov; Gavin Kelly; Becky Saunders; Michael Howell

    2012-01-01

    Oneogenic mutations in RAS genes are very common in human cancer,resulting in cells with well-characterized selective advantages,but also less well-understood vulnerabilities.We have carried out a large-scale loss-of-function screen to identify genes that are required by KRAS-transformed colon cancer cells,but not by derivatives lacking this oncogene.Top-scoring genes were then tested in a larger panel of KRAS mutant and wild-type cancer cells.Cancer cells expressing oncogenic KRAS were found to be highly dependent on the transcription factor GATA2 and the DNA replication initiation regulator CDC6.Extending this analysis using a collection of drugs with known targets,we found that cancer cells with mutant KRAS showed selective addiction to proteasome function,as well as synthetic lethality with topoisomerase inhibition.Combination targeting of these functions caused improved killing of KRAS mutant cells relative to wild-type cells.These observations suggest novel targets and new ways of combining existing therapies for optimal effect in RAS mutant cancers,which are traditionally seen as being highly refractory to therapy.

  1. Induction and selection of mutants from in vitro cultured plant cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yung Il; Kim, Jae Sung; Shin, In Chul; Lee, Sang Jae [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1994-07-01

    Mutant cell lines are useful for biochemical, physiological and genetical material for marker in various genetic manipulation experiments and for the direct use in crop plant improvement. Mutant selection may lead to the production of plants showing resistance or tolerance to specific environmental stress, such as solinity, drought, toxed metals, herbicides, pathogens and low temperature. In this review, these included the production of the somatic variation, the selection process itself and stability of the selected characters in cell culture and regenerated plant. Which would seem to be useful for improving plants and securring genetic resources. 45 refs. (Author).

  2. Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL) Reveals the Sequential Differentiation of Sieve Element-Like Cells.

    Science.gov (United States)

    Kondo, Yuki; Nurani, Alif Meem; Saito, Chieko; Ichihashi, Yasunori; Saito, Masato; Yamazaki, Kyoko; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Fukuda, Hiroo

    2016-06-01

    Cell differentiation is a complex process involving multiple steps, from initial cell fate specification to final differentiation. Procambial/cambial cells, which act as vascular stem cells, differentiate into both xylem and phloem cells during vascular development. Recent studies have identified regulatory cascades for xylem differentiation. However, the molecular mechanism underlying phloem differentiation is largely unexplored due to technical challenges. Here, we established an ectopic induction system for phloem differentiation named Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL). Our results verified similarities between VISUAL-induced Arabidopsis thaliana phloem cells and in vivo sieve elements. We performed network analysis using transcriptome data with VISUAL to dissect the processes underlying phloem differentiation, eventually identifying a factor involved in the regulation of the master transcription factor gene APL Thus, our culture system opens up new avenues not only for genetic studies of phloem differentiation, but also for future investigations of multidirectional differentiation from vascular stem cells. PMID:27194709

  3. Dissociative Ionization Mechanism and Appearance Energies in Adipic Acid Revealed by Imaging Photoelectron Photoion Coincidence, Selective Deuteration, and Calculations.

    Science.gov (United States)

    Heringa, Maarten F; Slowik, Jay G; Prévôt, André S H; Baltensperger, Urs; Hemberger, Patrick; Bodi, Andras

    2016-05-26

    Adipic acid, a model compound for oxygenated organic aerosol, has been studied at the VUV beamline of the Swiss Light Source. Internal energy selected cations were prepared by threshold photoionization using vacuum ultraviolet synchrotron radiation and imaging photoelectron photoion coincidence spectroscopy (iPEPICO). The threshold photoelectron spectrum yields a vertical ionization energy (IE) of 10.5 eV, significantly above the calculated adiabatic IE of 8.6 eV. The cationic minimum is accessible after vertical ionization by H-transfer from one of the γ-carbons to a carbonyl oxygen and is sufficiently energetic to decay by water loss at the ionization onset. The slope of the breakdown curves, quantum chemical calculations, and selective deuteration of the carboxylic hydrogens establish the dissociative photoionization mechanism. After ionization, one γ-methylene hydrogen and the two carboxylic hydrogens are randomized prior to H2O loss. On the basis of the deuteration degree in the H2O + CO-loss product at higher energies, a direct water-loss channel without complete randomization also exists. The breakdown diagram and center of gravity of the H2O + CO-loss peak were modeled to obtain 0 K appearance energies of 10.77, 10.32, and 11.53 eV for H2O + CO loss, CH2COOH loss, and H2O + CH2COOH loss from adipic acid. These agree well with the CBS-QB3 calculated values of 10.68, 10.45, and 11.57 eV, respectively, which shows that threshold photoionization can yield energetics data as long as the dissociation is statistical, even when the parent ion cannot be observed. The results can be used as a starting point for a deeper understanding of the ionization and low-energy fragmentation of organic aerosol components. PMID:27100102

  4. Glycoproteins of coated pits, cell junctions, and the entire cell surface revealed by monoclonal antibodies and immunoelectron microscopy

    OpenAIRE

    1983-01-01

    Topographical descriptions of three major plasma membrane glycoproteins of murine 3T3 cells were obtained by immunoelectron microscopy with monoclonal antibodies. A glycoprotein of Mr 80,000 was distributed throughout the total cell surface. A second of Mr 90,000 was concentrated in coated pits, and a third of Mr 100,000 was localized at cell junctions.

  5. Hypoxia selects bortezomib-resistant stem cells of chronic myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Michele Tanturli

    Full Text Available We previously demonstrated that severe hypoxia inhibits growth of Chronic Myeloid Leukemia (CML cells and selects stem cells where BCR/Abl(protein is suppressed, although mRNA is not, so that hypoxia-selected stem cells, while remaining leukemic, are independent of BCR/Abl signaling and thereby refractory to Imatinib-mesylate. The main target of this study was to address the effects of the proteasome inhibitor Bortezomib (BZ on the maintenance of stem or progenitor cells in hypoxic primary cultures (LC1, by determining the capacity of LC1 cells to repopulate normoxic secondary cultures (LC2 and the kinetics of this repopulation. Unselected K562 cells from day-2 hypoxic LC1 repopulated LC2 with rapid, progenitor-type kinetics; this repopulation was suppressed by BZ addition to LC1 at time 0, but completely resistant to day-1 BZ, indicating that progenitors require some time to adapt to stand hypoxia. K562 cells selected in hypoxic day-7 LC1 repopulated LC2 with stem-type kinetics, which was largely resistant to BZ added at either time 0 or day 1, indicating that hypoxia-selectable stem cells are BZ-resistant per se, i.e. before their selection. Furthermore, these cells were completely resistant to day-6 BZ, i.e. after selection. On the other hand, hypoxia-selected stem cells from CD34-positive cells of blast-crisis CML patients appeared completely resistant to either time-0 or day-1 BZ. To exploit in vitro the capacity of CML cells to adapt to hypoxia enabled to detect a subset of BZ-resistant leukemia stem cells, a finding of particular relevance in light of the fact that our experimental system mimics the physiologically hypoxic environment of bone marrow niches where leukemia stem cells most likely home and sustain minimal residual disease in vivo. This suggests the use of BZ as an enhanced strategy to control CML. in particular to prevent relapse of disease, to be considered with caution and to need further deepening.

  6. Selective effect of cell membrane on synaptic neurotransmission

    Science.gov (United States)

    Postila, Pekka A.; Vattulainen, Ilpo; Róg, Tomasz

    2016-01-01

    Atomistic molecular dynamics simulations were performed with 13 non-peptidic neurotransmitters (NTs) in three different membrane environments. The results provide compelling evidence that NTs are divided into membrane-binding and membrane-nonbinding molecules. NTs adhere to the postsynaptic membrane surface whenever the ligand-binding sites of their synaptic receptors are buried in the lipid bilayer. In contrast, NTs that have extracellular ligand-binding sites do not have a similar tendency to adhere to the membrane surface. This finding is a seemingly simple yet important addition to the paradigm of neurotransmission, essentially dividing it into membrane-independent and membrane-dependent mechanisms. Moreover, the simulations also indicate that the lipid composition especially in terms of charged lipids can affect the membrane partitioning of NTs. The revised paradigm, highlighting the importance of cell membrane and specific lipids for neurotransmission, should to be of interest to neuroscientists, drug industry and the general public alike.

  7. Selective effect of cell membrane on synaptic neurotransmission.

    Science.gov (United States)

    Postila, Pekka A; Vattulainen, Ilpo; Róg, Tomasz

    2016-01-01

    Atomistic molecular dynamics simulations were performed with 13 non-peptidic neurotransmitters (NTs) in three different membrane environments. The results provide compelling evidence that NTs are divided into membrane-binding and membrane-nonbinding molecules. NTs adhere to the postsynaptic membrane surface whenever the ligand-binding sites of their synaptic receptors are buried in the lipid bilayer. In contrast, NTs that have extracellular ligand-binding sites do not have a similar tendency to adhere to the membrane surface. This finding is a seemingly simple yet important addition to the paradigm of neurotransmission, essentially dividing it into membrane-independent and membrane-dependent mechanisms. Moreover, the simulations also indicate that the lipid composition especially in terms of charged lipids can affect the membrane partitioning of NTs. The revised paradigm, highlighting the importance of cell membrane and specific lipids for neurotransmission, should to be of interest to neuroscientists, drug industry and the general public alike. PMID:26782980

  8. 3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration

    Directory of Open Access Journals (Sweden)

    Jyuhn-Huarng Juang

    2015-02-01

    Full Text Available The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histology with cell tracing to reveal the participation of Schwann cells and pericytes in mouse islet transplantation. Longitudinal studies of the grafts under the kidney capsule identify that the donor Schwann cells and pericytes re-associate with the engrafted islets at the peri-graft and perivascular domains, respectively, indicating their adaptability in transplantation. Based on the morphological proximity and cellular reactivity, we propose that the new islet microenvironment should include the peri-graft Schwann cell sheath and perivascular pericytes as an integral part of the new tissue.

  9. NACA deficiency reveals the crucial role of somite-derived stromal cells in haematopoietic niche formation.

    Science.gov (United States)

    Murayama, Emi; Sarris, Milka; Redd, Michael; Le Guyader, Dorothée; Vivier, Catherine; Horsley, Wyatt; Trede, Nikolaus; Herbomel, Philippe

    2015-01-01

    The ontogeny of haematopoietic niches in vertebrates is essentially unknown. Here we show that the stromal cells of the caudal haematopoietic tissue (CHT), the first niche where definitive haematopoietic stem/progenitor cells (HSPCs) home in zebrafish development, derive from the caudal somites through an epithelial-mesenchymal transition (EMT). The resulting stromal cell progenitors accompany the formation of the caudal vein sinusoids, the other main component of the CHT niche, and mature into reticular cells lining and interconnecting sinusoids. We characterize a zebrafish mutant defective in definitive haematopoiesis due to a deficiency in the nascent polypeptide-associated complex alpha subunit (NACA). We demonstrate that the defect resides not in HSPCs but in the CHT niche. NACA-deficient stromal cell progenitors initially develop normally together with the sinusoids, and HSPCs home to the resulting niche, but stromal cell maturation is compromised, leading to a niche that is unable to support HSPC maintenance, expansion and differentiation. PMID:26411530

  10. Autonomy and Non-autonomy of Angiogenic Cell Movements Revealed by Experiment-Driven Mathematical Modeling

    Directory of Open Access Journals (Sweden)

    Kei Sugihara

    2015-12-01

    Full Text Available Angiogenesis is a multicellular phenomenon driven by morphogenetic cell movements. We recently reported morphogenetic vascular endothelial cell (EC behaviors to be dynamic and complex. However, the principal mechanisms orchestrating individual EC movements in angiogenic morphogenesis remain largely unknown. Here we present an experiment-driven mathematical model that enables us to systematically dissect cellular mechanisms in branch elongation. We found that cell-autonomous and coordinated actions governed these multicellular behaviors, and a cell-autonomous process sufficiently illustrated essential features of the morphogenetic EC dynamics at both the single-cell and cell-population levels. Through refining our model and experimental verification, we further identified a coordinated mode of tip EC behaviors regulated via a spatial relationship between tip and follower ECs, which facilitates the forward motility of tip ECs. These findings provide insights that enhance our mechanistic understanding of not only angiogenic morphogenesis, but also other types of multicellular phenomenon.

  11. Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis.

    Science.gov (United States)

    Shimada, Kenichi; Skouta, Rachid; Kaplan, Anna; Yang, Wan Seok; Hayano, Miki; Dixon, Scott J; Brown, Lewis M; Valenzuela, Carlos A; Wolpaw, Adam J; Stockwell, Brent R

    2016-07-01

    Apoptosis is one type of programmed cell death. Increasingly, non-apoptotic cell death is recognized as being genetically controlled, or 'regulated'. However, the full extent and diversity of alternative cell death mechanisms remain uncharted. Here we surveyed the landscape of pharmacologically accessible cell death mechanisms. In an examination of 56 caspase-independent lethal compounds, modulatory profiling showed that 10 compounds induced three different types of regulated non-apoptotic cell death. Optimization of one of those ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis has been found to occur when the lipid-repair enzyme GPX4 is inhibited. FIN56 promoted degradation of GPX4. FIN56 also bound to and activated squalene synthase, an enzyme involved in isoprenoid biosynthesis, independent of GPX4 degradation. These discoveries show that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes. PMID:27159577

  12. Fine tuning of the threshold of T cell selection by the Nck adapters.

    Science.gov (United States)

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Schmitt, Sabine; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter J; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-12-15

    Thymic selection shapes the T cell repertoire to ensure maximal antigenic coverage against pathogens while preventing autoimmunity. Recognition of self-peptides in the context of peptide-MHC complexes by the TCR is central to this process, which remains partially understood at the molecular level. In this study we provide genetic evidence that the Nck adapter proteins are essential for thymic selection. In vivo Nck deletion resulted in a reduction of the thymic cellularity, defective positive selection of low-avidity T cells, and impaired deletion of thymocytes engaged by low-potency stimuli. Nck-deficient thymocytes were characterized by reduced ERK activation, particularly pronounced in mature single positive thymocytes. Taken together, our findings identify a crucial role for the Nck adapters in enhancing TCR signal strength, thereby fine-tuning the threshold of thymocyte selection and shaping the preimmune T cell repertoire. PMID:21078909

  13. Effects of Paclitaxel on EGFR Endocytic Trafficking Revealed Using Quantum Dot Tracking in Single Cells

    OpenAIRE

    Li, Hui; Duan, Zhao-Wen; Xie, Ping; Liu, Yu-Ru; Wang, Wei-Chi; Dou, Shuo-Xing; Wang, Peng-Ye

    2012-01-01

    Paclitaxel (PTX), a chemotherapeutic drug, affects microtubule dynamics and influences endocytic trafficking. However, the mechanism and the dynamics of altered endocytic trafficking by paclitaxel treatment in single living cells still remain elusive. By labeling quantum dots (QDs) to the epidermal growth factor (EGF), we continuously tracked the endocytosis and post-endocytic trafficking of EGF receptors (EGFRs) in A549 cells for a long time interval. A single-cell analysis method was introd...

  14. Lineage tracing reveals the dynamic contribution of Hes1+ cells to the developing and adult pancreas

    OpenAIRE

    Kopinke, Daniel; Brailsford, Marisa; Shea, Jill E; Leavitt, Rebecca; Scaife, Courtney L.; Murtaugh, L. Charles

    2011-01-01

    Notch signaling regulates numerous developmental processes, often acting either to promote one cell fate over another or else to inhibit differentiation altogether. In the embryonic pancreas, Notch and its target gene Hes1 are thought to inhibit endocrine and exocrine specification. Although differentiated cells appear to downregulate Hes1, it is unknown whether Hes1 expression marks multipotent progenitors, or else lineage-restricted precursors. Moreover, although rare cells of the adult pan...

  15. Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria

    OpenAIRE

    Gefen, Orit; Gabay, Chana; Mumcuoglu, Michael; Engel, Giora; Balaban, Nathalie Q.

    2008-01-01

    Phenotypic variability in populations of cells has been linked to evolutionary robustness to stressful conditions. A remarkable example of the importance of cell-to-cell variability is found in bacterial persistence, where subpopulations of dormant bacteria, termed persisters, were shown to be responsible for the persistence of the population to antibiotic treatments. Here, we use microfluidic devices to monitor the induction of fluorescent proteins under synthetic promoters and characterize ...

  16. Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

    International Nuclear Information System (INIS)

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed

  17. Tissue- and cell-specific functions of the androgen receptor revealed through conditional knockout models in mice.

    Science.gov (United States)

    De Gendt, Karel; Verhoeven, Guido

    2012-04-16

    This review aims to evaluate the contribution of individual cell-selective knockout models to our current understanding of androgen action. Cre/loxP technology has allowed the generation of cell-selective knockout models targeting the androgen receptor (AR) in distinct putative target cells in a wide variety of organs and tissues including: testis, ovary, accessory sex tissues, muscle, bone, fat, liver, skin and myeloid tissue. In some androgen-regulated processes such as spermatogenesis and folliculogenesis this approach has lead to the identification of a key cellular mediator of androgen action (Sertoli and granulosa cells, respectively). In many target tissues, however, the final response to androgens appears to be more complex. Here, cell-selective knockout technology offers a platform upon which we can begin to unravel the more complex interplay and signaling pathways of androgens. A prototypic example is the analysis of mesenchymal-epithelial interactions in many accessory sex glands. Furthermore, for some actions of testosterone, in which part of the effect is mediated by the active metabolite 17β-estradiol, conditional knockout technology offers a novel strategy to study the relative contribution of AR and estrogen receptor-mediated signaling. The latter approach has already resulted in a better understanding of androgen action in brain and bone. Finally, cell-selective knockout technology has generated valuable models to search for AR-controlled molecular mediators of androgen action, a strategy that has successfully been applied to the study of androgen action in the testis and in the epididymis. Although some conditional knockout models have provided clear answers to physiologic questions, it should be noted that others have pointed to unexpected complexities or technical limitations confounding interpretation of the results. PMID:21871526

  18. Selective Retention of Bone Marrow-Derived Cells to Enhance Spinal Fusion

    OpenAIRE

    Muschler, George F.; Matsukura, Yoichi; Nitto, Hironori; Boehm, Cynthia A.; Valdevit, Antonio D.; Kambic, Helen E.; Davros, William J.; Easley, Kirk A.; Powell, Kimerly A.

    2005-01-01

    Connective tissue progenitors can be concentrated rapidly from fresh bone marrow aspirates using some porous matrices as a surface for cell attachment and selective retention, and for creating a cellular graft that is enriched with respect to the number of progenitor cells. We evaluated the potential value of this method using demineralized cortical bone powder as the matrix. Matrix alone, matrix plus marrow, and matrix enriched with marrow cells were compared in an established canine spinal ...

  19. Black silicon laser-doped selective emitter solar cell with 18.1% efficiency

    DEFF Research Database (Denmark)

    Davidsen, Rasmus Schmidt; Li, Hongzhao; To, Alexander;

    2016-01-01

    We report fabrication of nanostructured, laser-doped selective emitter (LDSE) silicon solar cells with power conversion efficiency of 18.1% and a fill factor (FF) of 80.1%. The nanostructured solar cells were realized through a single step, mask-less, scalable reactive ion etch (RIE) texturing......-texturing as well as the LDSE process, we consider this specific combination a promising candidate for a cost-efficient process for future Si solar cells....

  20. Kinome sequencing reveals RET G691S polymorphism in human neuroendocrine lung cancer cell lines

    OpenAIRE

    Sosonkina, Nadiya; HONG, SEUNG-KEUN; Starenki, Dmytro; Park, Jong-In

    2014-01-01

    Neuroendocrine (NE) lung tumors comprise 20–25% of all invasive lung malignancies. Currently, no effective treatments are available to cure these tumors, and it is necessary to identify a molecular alteration(s) that characterizes NE lung tumor cells. We aimed to identify a kinase mutation(s) associated with NE lung tumor by screening 517 kinase-encoding genes in human lung cancer cell lines. Our next-generation sequencing analysis of six NE lung tumor cell lines (four small cell lung cancer ...

  1. Nanotube action between human mesothelial cells reveals novel aspects of inflammatory responses.

    Directory of Open Access Journals (Sweden)

    Julia Ranzinger

    Full Text Available A well-known role of human peritoneal mesothelial cells (HPMCs, the resident cells of the peritoneal cavity, is the generation of an immune response during peritonitis by activation of T-cells via antigen presentation. Recent findings have shown that intercellular nanotubes (NTs mediate functional connectivity between various cell types including immune cells - such as T-cells, natural killer (NK cells or macrophages - by facilitating a spectrum of long range cell-cell interactions. Although of medical interest, the relevance of NT-related findings for human medical conditions and treatment, e.g. in relation to inflammatory processes, remains elusive, particularly due to a lack of appropriate in vivo data. Here, we show for the first time that primary cultures of patient derived HPMCs are functionally connected via membranous nanotubes. NT formation appears to be actin cytoskeleton dependent, mediated by the action of filopodia. Importantly, significant variances in NT numbers between different donors as a consequence of pathophysiological alterations were observable. Furthermore, we show that TNF-α induces nanotube formation and demonstrate a strong correlation of NT connectivity in accordance with the cellular cholesterol level and distribution, pointing to a complex involvement of NTs in inflammatory processes with potential impact for clinical treatment.

  2. Selective, rapid and optically switchable regulation of protein function in live mammalian cells

    Science.gov (United States)

    Tsai, Yu-Hsuan; Essig, Sebastian; James, John R.; Lang, Kathrin; Chin, Jason W.

    2015-07-01

    The rapid and selective regulation of a target protein within living cells that contain closely related family members is an outstanding challenge. Here we introduce genetically directed bioorthogonal ligand tethering (BOLT) and demonstrate selective inhibition (iBOLT) of protein function. In iBOLT, inhibitor-conjugate/target protein pairs are created where the target protein contains a genetically encoded unnatural amino acid with bioorthogonal reactivity and the inhibitor conjugate contains a complementary bioorthogonal group. iBOLT enables the first rapid and specific inhibition of MEK isozymes, and introducing photoisomerizable linkers in the inhibitor conjugate enables reversible, optical regulation of protein activity (photo-BOLT) in live mammalian cells. We demonstrate that a pan kinase inhibitor conjugate allows selective and rapid inhibition of the lymphocyte specific kinase, indicating the modularity and scalability of BOLT. We anticipate that BOLT will enable the rapid and selective regulation of diverse proteins for which no selective small-molecule ligands exist.

  3. Selective loss of B-cell phenotype in lymphocyte predominant Hodgkin lymphoma.

    Science.gov (United States)

    Tedoldi, S; Mottok, A; Ying, J; Paterson, J C; Cui, Y; Facchetti, F; van Krieken, J H J M; Ponzoni, M; Ozkal, S; Masir, N; Natkunam, Y; Pileri, Sa; Hansmann, M-L; Mason, Dy; Tao, Q; Marafioti, T

    2007-12-01

    The neoplastic Reed-Sternberg cells characteristic of classical Hodgkin's lymphoma (cHL) are of B-cell origin but they almost always show striking loss of a range of B-cell-associated molecules. In contrast, the neoplastic cells found in lymphocyte predominant Hodgkin's lymphoma (LPHL) (L&H cells) are traditionally thought of as possessing the full repertoire of features associated with germinal centre B cells (eg BCL-6 expression, 'ongoing' Ig gene mutation). In the present paper, we report an extensive phenotypic analysis of L&H cells which revealed down-regulation of a number of markers associated with the B-cell lineage (eg CD19, CD37) and with the germinal centre maturation stage (eg PAG, LCK). The promoter methylation status of three of these down-regulated genes (CD10, CD19, and LCK) was further studied in microdissected L&H cells, and this revealed that their promoters were unmethylated. In contrast, these genes showed promoter methylation in cell lines derived from CHL. Further investigation of the mechanisms responsible for the deregulation of these molecules in L&H cells may provide new insights into the genetic abnormalities underlying LPHL. PMID:17935142

  4. Coupling of HIV-1 Antigen to the Selective Autophagy Receptor SQSTM1/p62 Promotes T-Cell-Mediated Immunity

    Science.gov (United States)

    Andersen, Aram Nikolai; Landsverk, Ole Jørgen; Simonsen, Anne; Bogen, Bjarne; Corthay, Alexandre; Øynebråten, Inger

    2016-01-01

    Vaccines aiming to promote T-cell-mediated immune responses have so far showed limited efficacy, and there is a need for novel strategies. Studies indicate that autophagy plays an inherent role in antigen processing and presentation for CD4+ and CD8+ T cells. Here, we report a novel vaccine strategy based on fusion of antigen to the selective autophagy receptor sequestosome 1 (SQSTM1)/p62. We hypothesized that redirection of vaccine antigen from proteasomal degradation into the autophagy pathway would increase the generation of antigen-specific T cells. A hybrid vaccine construct was designed in which the antigen is fused to the C-terminus of p62, a signaling hub, and a receptor that naturally delivers ubiquitinated cargo for autophagic degradation. Fusion of the human immunodeficiency virus-1 antigen Gagp24 to p62 resulted in efficient antigen delivery into the autophagy pathway. Intradermal immunization of mice revealed that, in comparison to Gagp24 delivered alone, fusion to p62 enhanced the number of Gagp24-specific interferon-γ-producing T cells, including CD8+ T cells. The strategy may also have the potential to modulate the antigenic peptide repertoire. Because p62 and autophagy are highly conserved between species, we anticipate this strategy to be a candidate for the development of T-cell-based vaccines in humans.

  5. Optimization of Cat's Whiskers Tea (Orthosiphon stamineus) Using Supercritical Carbon Dioxide and Selective Chemotherapeutic Potential against Prostate Cancer Cells.

    Science.gov (United States)

    Al-Suede, Fouad Saleih R; Khadeer Ahamed, Mohamed B; Abdul Majid, Aman S; Baharetha, Hussin M; Hassan, Loiy E A; Kadir, Mohd Omar A; Nassar, Zeyad D; Abdul Majid, Amin M S

    2014-01-01

    Cat's whiskers (Orthosiphon stamineus) leaves extracts were prepared using supercritical CO2 (SC-CO2) with full factorial design to determine the optimum extraction parameters. Nine extracts were obtained by varying pressure, temperature, and time. The extracts were analysed using FTIR, UV-Vis, and GC-MS. Cytotoxicity of the extracts was evaluated on human (colorectal, breast, and prostate) cancer and normal fibroblast cells. Moderate pressure (31.1 MPa) and temperature (60°C) were recorded as optimum extraction conditions with high yield (1.74%) of the extract (B2) at 60 min extraction time. The optimized extract (B2) displayed selective cytotoxicity against prostate cancer (PC3) cells (IC50 28 µg/mL) and significant antioxidant activity (IC50 42.8 µg/mL). Elevated levels of caspases 3/7 and 9 in B2-treated PC3 cells suggest the induction of apoptosis through nuclear and mitochondrial pathways. Hoechst and rhodamine assays confirmed the nuclear condensation and disruption of mitochondrial membrane potential in the cells. B2 also demonstrated inhibitory effects on motility and colonies of PC3 cells at its subcytotoxic concentrations. It is noteworthy that B2 displayed negligible toxicity against the normal cells. Chemometric analysis revealed high content of essential oils, hydrocarbon, fatty acids, esters, and aromatic sesquiterpenes in B2. This study highlights the therapeutic potentials of SC-CO2 extract of cat's whiskers in targeting prostate carcinoma. PMID:25276215

  6. Low concentrations of metformin selectively inhibit CD133⁺ cell proliferation in pancreatic cancer and have anticancer action.

    Science.gov (United States)

    Gou, Shanmiao; Cui, Pengfei; Li, Xiangsheng; Shi, Pengfei; Liu, Tao; Wang, Chunyou

    2013-01-01

    Pancreatic cancer is the fourth leading cause of cancer related deaths in the United States. The prognosis remains dismal with little advance in treatment. Metformin is a drug widely used for the treatment of type II diabetes. Recent epidemiologic data revealed that oral administration of metformin is associated with a reduced risk of pancreatic cancer, suggesting its potential as a novel drug for this disease. Many studies have demonstrated the in vitro anticancer action of metformin, but the typically used concentrations were much higher than the in vivo plasma and tissue concentrations achieved with recommended therapeutic doses of metformin, and low concentrations of metformin had little effect on the proliferation of pancreatic cancer cells. We examined the effect of low concentrations of metformin on different subpopulations of pancreatic cancer cells and found that these selectively inhibited the proliferation of CD133⁺ but not CD24⁺CD44⁺ESA⁺ cells. We also examined the effect of low concentrations of metformin on cell invasion and in vivo tumor formation, demonstrating in vitro and in vivo anticancer action. Metformin was associated with a reduction of phospho-Erk and phospho-mTOR independent of Akt and AMPK phosphorylation. CD133⁺ pancreatic cancer cells are considered to be cancer stem cells that contribute to recurrence, metastasis and resistance to adjuvant therapies in pancreatic cancer. Our results provide a basis for combination of metformin with current therapies to improve the prognosis of this disease. PMID:23667692

  7. Development of a highly metastatic model that reveals a crucial role of fibronectin in lung cancer cell migration and invasion

    International Nuclear Information System (INIS)

    The formation of metastasis is the most common cause of death in patients with lung cancer. A major implement to understand the molecular mechanisms involved in lung cancer metastasis has been the lack of suitable models to address it. In this study, we aimed at establishing a highly metastatic model of human lung cancer and characterizing its metastatic properties and underlying mechanisms. The human lung adeno-carcinoma SPC-A-1 cell line was used as parental cells for developing of highly metastatic cells by in vivo selection in NOD/SCID mice. After three rounds of selection, a new SPC-A-1sci cell line was established from pulmonary metastatic lesions. Subsequently, the metastatic properties of this cell line were analyzed, including optical imaging of in vivo metastasis, immunofluorescence and immunohistochemical analysis of several epithelial mesenchymal transition (EMT) makers and trans-well migration and invasion assays. Finally, the functional roles of fibronectin in the invasive and metastatic potentials of SPC-A-1sci cells were determined by shRNA analysis. A spontaneously pulmonary metastatic model of human lung adeno-carcinoma was established in NOD/SCID mice, from which a new lung cancer cell line, designated SPC-A-1sci, was isolated. Initially, the highly metastatic behavior of this cell line was validated by optical imaging in mice models. Further analyses showed that this cell line exhibit phenotypic and molecular alterations consistent with EMT. Compared with its parent cell line SPC-A-1, SPC-A-1sci was more aggressive in vitro, including increased potentials for cell spreading, migration and invasion. Importantly, fibronectin, a mesenchymal maker of EMT, was found to be highly expressed in SPC-A-1sci cells and down-regulation of it can decrease the in vitro and in vivo metastatic abilities of this cell line. We have successfully established a new human lung cancer cell line with highly metastatic potentials, which is subject to EMT and possibly

  8. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity.

    Science.gov (United States)

    Noutsi, Pakiza; Gratton, Enrico; Chaieb, Sahraoui

    2016-01-01

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines. PMID:27362860

  9. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity

    KAUST Repository

    Noutsi, Pakiza

    2016-06-30

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  10. Time course of morphine's effects on adult hippocampal subgranular zone reveals preferential inhibition of cells in S phase of the cell cycle and a subpopulation of immature neurons.

    Science.gov (United States)

    Arguello, A A; Harburg, G C; Schonborn, J R; Mandyam, C D; Yamaguchi, M; Eisch, A J

    2008-11-11

    Opiates, such as morphine, decrease neurogenesis in the adult hippocampal subgranular zone (SGZ), raising the possibility that decreased neurogenesis contributes to opiate-induced cognitive deficits. However, there is an incomplete understanding of how alterations in cell cycle progression and progenitor maturation contribute to this decrease. The present study examined how morphine regulates progenitor cell cycle, cell death and immature SGZ neurons (experiment 1) as well as the progression of SGZ progenitors through key stages of maturation (experiment 2). In experiment 1, mice received sham or morphine pellets (s.c., 0 and 48 h) and bromodeoxyuridine (BrdU) 2 h prior to sacrifice (24, 72 or 96 h). Morphine decreased both the number of S phase and total cycling cells, as there were fewer cells immunoreactive (IR) for the S phase marker BrdU and the cell cycle marker Ki67. The percentage of Ki67-IR cells that were BrdU-IR was decreased after 24 but not 96 h of morphine, suggesting a disproportionate effect on S phase cells relative to all cycling cells at this time point. Cell death (activated caspase-3 counts) was increased after 24 but not 96 h. In experiment 2, nestin-green fluorescent protein (GFP) mice given BrdU 1 day prior to morphine or sham surgery (0 and 48 h, sacrifice 96 h) had fewer Ki67-IR cells, but no change in BrdU-IR cell number, suggesting that this population of BrdU-IR cells was less sensitive to morphine. Interestingly, examination of key stages of progenitor cell maturation revealed that morphine increased the percent of BrdU-IR cells that were type 2b and decreased the percent that were immature neurons. These data suggest that chronic morphine decreases SGZ neurogenesis by inhibiting dividing cells, particularly those in S phase, and progenitor cell progression to a more mature neuronal stage. PMID:18832014

  11. Live imaging-based model selection reveals periodic regulation of the stochastic G1/S phase transition in vertebrate axial development.

    Science.gov (United States)

    Sugiyama, Mayu; Saitou, Takashi; Kurokawa, Hiroshi; Sakaue-Sawano, Asako; Imamura, Takeshi; Miyawaki, Atsushi; Iimura, Tadahiro

    2014-12-01

    In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1) fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of the

  12. Live imaging-based model selection reveals periodic regulation of the stochastic G1/S phase transition in vertebrate axial development.

    Directory of Open Access Journals (Sweden)

    Mayu Sugiyama

    2014-12-01

    Full Text Available In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1 fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1 notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of

  13. Modern genome-wide genetic approaches to reveal intrinsic properties of stem cells

    NARCIS (Netherlands)

    de Haan, Gerald; Gerrits, Alice; Bystrykh, Leonid

    2006-01-01

    Purpose of review The clinical use of hematopoietic stem cells, which produce all mature blood cell lineages in the circulation, is continuously increasing. Identification of genes and gene networks specifying either sternness or commitment will not only be of major relevance for a fundamental under

  14. High-resolution telomere fluorescence in situ hybridization reveals intriguing anomalies in germ cell tumors.

    Science.gov (United States)

    Shekhani, Mohammed Talha; Barber, John R; Bezerra, Stephania M; Heaphy, Christopher M; Gonzalez Roibon, Nilda Diana; Taheri, Diana; Reis, Leonardo O; Guner, Gunes; Joshu, Corinne E; Netto, George J; Meeker, Alan K

    2016-08-01

    Testicular germ cell tumor (TGCT) is the most common malignancy of young men. Most patients are completely cured, which distinguishes these from most other malignancies. Orchiectomy specimens (n=76) were evaluated using high-resolution (single-cell discriminative) telomere-specific fluorescence in situ hybridization (FISH) with simultaneous Oct4 immunofluorescence to describe telomere length phenotype in TGCT neoplastic cells. For the first time, the TGCT precursor lesion, germ cell neoplasia in situ (GCNIS) is also evaluated in depth. The intensity of the signals from cancerous cells was compared to the same patient's reference cells-namely, healthy germ cells (defined as "medium" length) and interstitial/somatic cells (defined as "short" telomere length). We observed short telomeres in most GCNIS and pure seminomas (P=.006 and P=.0005, respectively). In contrast, nonseminomas displayed longer telomeres. Lesion-specific telomere lengths were documented in mixed tumor cases. Embryonal carcinoma (EC) demonstrated the longest telomeres. A fraction of EC displays the telomerase-independent alternative lengthening of telomeres (ALT) phenotype (24% of cases). Loss of ATRX or DAXX nuclear expression was strongly associated with ALT; however, nuclear expression of both proteins was retained in half of ALT-positive ECs. The particular distribution of telomere lengths among TGCT and GCNIS precursors implicate telomeres anomalies in pathogenesis. These results may advise management decisions as well. PMID:27085557

  15. Gene expression profiling revealed novel mechanism of action of Taxotere and Furtulon in prostate cancer cells

    International Nuclear Information System (INIS)

    Both Taxotere and Capecitabine have shown anti-cancer activity against various cancers including prostate cancer. In combination, Taxotere plus Capecitabine has demonstrated higher anti-cancer activity in advanced breast cancers. However, the molecular mechanisms of action of Taxotere and Capecitabine have not been fully elucidated in prostate cancer. The total RNA from PC3 and LNCaP prostate cells untreated and treated with 2 nM Taxotere, 110 μM Furtulon (active metabolite of Capecitabine), or 1 nM Taxotere plus 50 μM Furtulon for 6, 36, and 72 hours, was subjected to Affymetrix Human Genome U133A Array analysis. Real-time PCR and Western Blot analysis were conducted to confirm microarray data. Taxotere and Furtulon down-regulated some genes critical for cell proliferation, cell cycle progression, transcription factor, cell signaling, and oncogenesis, and up-regulated some genes related to the induction of apoptosis, cell cycle arrest, and differentiation in both cell lines. Taxotere and Furtulon also up-regulated some genes responsible for chemotherapeutic resistance, suggesting the induction of cancer cell resistance to these agents. Taxotere and Furtulon caused the alternation of a large number of genes, many of which may contribute to the molecular mechanisms by which Taxotere and Furtulon inhibit the growth of prostate cancer cells. This information could be utilized for further mechanistic research and for devising optimized therapeutic strategies against prostate cancer

  16. Targeted cell elimination reveals an auxin-guided biphasic mode of lateral root initiation

    Science.gov (United States)

    Marhavý, Peter; Montesinos, Juan Carlos; Abuzeineh, Anas; Van Damme, Daniel; Vermeer, Joop E.M.; Duclercq, Jerôme; Rakusová, Hana; Nováková, Petra; Friml, Jiři; Geldner, Niko; Benková, Eva

    2016-01-01

    To sustain a lifelong ability to initiate organs, plants retain pools of undifferentiated cells with a preserved proliferation capacity. The root pericycle represents a unique tissue with conditional meristematic activity, and its tight control determines initiation of lateral organs. Here we show that the meristematic activity of the pericycle is constrained by the interaction with the adjacent endodermis. Release of these restraints by elimination of endodermal cells by single-cell ablation triggers the pericycle to re-enter the cell cycle. We found that endodermis removal substitutes for the phytohormone auxin-dependent initiation of the pericycle meristematic activity. However, auxin is indispensable to steer the cell division plane orientation of new organ-defining divisions. We propose a dual, spatiotemporally distinct role for auxin during lateral root initiation. In the endodermis, auxin releases constraints arising from cell-to-cell interactions that compromise the pericycle meristematic activity, whereas, in the pericycle, auxin defines the orientation of the cell division plane to initiate lateral roots. PMID:26883363

  17. Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem cells

    DEFF Research Database (Denmark)

    Pines, Alex; Kelstrup, Christian D; Vrouwe, Mischa G;

    2011-01-01

    (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia...

  18. Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

    Directory of Open Access Journals (Sweden)

    Yevgeniy A Grigoryev

    Full Text Available A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+CD62L(- effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

  19. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

    DEFF Research Database (Denmark)

    Lewis, Nathan E; Liu, Xin; Li, Yuxiang; Nagarajan, Harish; Yerganian, George; O'Brien, Edward; Bordbar, Aarash; Roth, Anne M; Rosenbloom, Jeffrey; Bian, Chao; Xie, Min; Chen, Wenbin; Li, Ning; Baycin-Hizal, Deniz; Latif, Haythem; Förster, Jochen; Betenbaugh, Michael; Famili, Iman; Xu, Xun; Wang, Jun; Palsson, Bernhard O

    2013-01-01

    Chinese hamster ovary (CHO) cells, first isolated in 1957, are the preferred production host for many therapeutic proteins. Although genetic heterogeneity among CHO cell lines has been well documented, a systematic, nucleotide-resolution characterization of their genotypic differences has been...... stymied by the lack of a unifying genomic resource for CHO cells. Here we report a 2.4-Gb draft genome sequence of a female Chinese hamster, Cricetulus griseus, harboring 24,044 genes. We also resequenced and analyzed the genomes of six CHO cell lines from the CHO-K1, DG44 and CHO-S lineages. This...... analysis identified hamster genes missing in different CHO cell lines, and detected >3.7 million single-nucleotide polymorphisms (SNPs), 551,240 indels and 7,063 copy number variations. Many mutations are located in genes with functions relevant to bioprocessing, such as apoptosis. The details of this...

  20. Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Vancini, Ricardo [Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC (United States); Kramer, Laura D. [Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, Albany, NY (United States); Ribeiro, Mariana; Hernandez, Raquel [Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC (United States); Brown, Dennis, E-mail: dennis_brown@ncsu.edu [Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC (United States)

    2013-01-20

    Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.

  1. Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane

    International Nuclear Information System (INIS)

    Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.

  2. β-Catenin Inhibits T Cell Activation by Selective Interference with Linker for Activation of T Cells–Phospholipase C-γ1 Phosphorylation

    Science.gov (United States)

    Driessens, Gregory; Zheng, Yan; Locke, Frederick; Cannon, Judy L.; Gounari, Fotini; Gajewski, Thomas F.

    2016-01-01

    Despite the defined function of the β-catenin pathway in thymocytes, its functional role in peripheral T cells is poorly understood. We report that in a mouse model, β-catenin protein is constitutively degraded in peripheral T cells. Introduction of stabilized β-catenin into primary T cells inhibited proliferation and cytokine secretion after TCR stimulation and blunted effector cell differentiation. Functional and biochemical studies revealed that β-catenin selectively inhibited linker for activation of T cells phosphorylation on tyrosine 136, which was associated with defective phospholipase C-γ1 phosphorylation and calcium signaling but normal ERK activation. Our findings indicate that β-catenin negatively regulates T cell activation by a previously undescribed mechanism and suggest that conditions under which β-catenin might be inducibly stabilized in vivo would be inhibitory for T cell-based immunity. PMID:21149602

  3. Genetic diversity analysis reveals that geographical environment plays a more important role than rice cultivar in Villosiclava virens population selection.

    Science.gov (United States)

    Wang, Fei; Zhang, Shu; Liu, Mei-Gang; Lin, Xian-Song; Liu, Hui-Jiang; Peng, You-Liang; Lin, Yang; Huang, Jun-Bin; Luo, Chao-Xi

    2014-05-01

    Rice false smut caused by Villosiclava virens is an economically important disease of grains worldwide. The genetic diversity of 153 isolates from six fields located in Wuhan (WH), Yichang Wangjia (YCW), Yichang Yaohe (YCY), Huanggang (HG), Yangxin (YX), and Jingzhou (JZ) in Hubei province of China were phylogenetically analyzed to evaluate the influence of environments and rice cultivars on the V. virens populations. Isolates (43) from Wuhan were from two rice cultivars, Wanxian 98 and Huajing 952, while most of the other isolates from fields YCW, YCY, HG, YX, and JZ originated from different rice cultivars with different genetic backgrounds. Genetic diversity of isolates was analyzed using random amplified polymorphic DNA (RAPD) and single-nucleotide polymorphisms (SNP). The isolates from the same cultivars in Wuhan tended to group together, indicating that the cultivars had an important impact on the fungal population. The 110 isolates from individual fields tended to cluster according to geographical origin. The values of Nei's gene diversity (H) and Shannon's information index (I) showed that the genetic diversity among isolates was higher between than within geographical populations. Furthermore, mean genetic distance between groups (0.006) was higher than mean genetic distance within groups (0.0048) according to MEGA 5.2. The pairwise population fixation index (FST) values also showed significant genetic differentiation between most populations. Higher genetic similarity of isolates from individual fields but different rice cultivars suggested that the geographical factor played a more important role in the selection of V. virens isolates than rice cultivars. This information could be used to improve the management strategy for rice false smut by adjusting the cultivation measures, such as controlling fertilizer, water, and planting density, in the rice field to change the microenvironment. PMID:24584249

  4. Genetic differentiation revealed by selective loci of drought-responding EST-SSRs between upland and lowland rice in China.

    Directory of Open Access Journals (Sweden)

    Hui Xia

    Full Text Available Upland and lowland rice (Oryza sativa L. represent two of the most important rice ecotypes adapted to ago-ecosystems with contrasting soil-water conditions. Upland rice, domesticated in the water-limited environment, contains valuable drought-resistant characters that can be used in water-saving breeding. Knowledge about the divergence between upland and lowland rice will provide valuable cues for the evolution of drought-resistance in rice. Genetic differentiation between upland and lowland rice was explored by 47 Simple Sequence Repeats (SSRs located in drought responding expressed sequence tags (ESTs among 377 rice landraces. The morphological traits of drought-resistance were evaluated in the field experiments. Different outlier loci were detected in the japonica and indica subspecies, respectively. Considerable genetic differentiation between upland and lowland rice on these outlier loci was estimated in japonica (Fst = 0.258 and indica (Fst = 0.127. Furthermore, populations of the upland and lowland ecotypes were clustered separately on these outlier loci. A significant correlation between genetic distance matrices and the dissimilarity matrices of drought-resistant traits was determined, indicating a certain relationship between the upland-lowland rice differentiation and the drought-resistance. Divergent selections occur between upland and lowland rice on the drought-resistance as the Qsts of some drought-resistant traits are significantly higher than the neutral Fst. In addition, the upland- and lowland-preferable alleles responded differently among ecotypes or allelic types under osmotic stress. This shows the evolutionary signature of drought resistance at the gene expression level. The findings of this study can strengthen our understanding of the evolution of drought-resistance in rice with significant implications in the improvement of rice drought-resistance.

  5. Chromosomal copy number variation, selection and uneven rates of recombination reveal cryptic genome diversity linked to pathogenicity.

    Directory of Open Access Journals (Sweden)

    Rhys A Farrer

    Full Text Available Pathogenic fungi constitute a growing threat to both plant and animal species on a global scale. Despite a clonal mode of reproduction dominating the population genetic structure of many fungi, putatively asexual species are known to adapt rapidly when confronted by efforts to control their growth and transmission. However, the mechanisms by which adaptive diversity is generated across a clonal background are often poorly understood. We sequenced a global panel of the emergent amphibian pathogen, Batrachochytrium dendrobatidis (Bd, to high depth and characterized rapidly changing features of its genome that we believe hold the key to the worldwide success of this organism. Our analyses show three processes that contribute to the generation of de novo diversity. Firstly, we show that the majority of wild isolates manifest chromosomal copy number variation that changes over short timescales. Secondly, we show that cryptic recombination occurs within all lineages of Bd, leading to large regions of the genome being in linkage equilibrium, and is preferentially associated with classes of genes of known importance for virulence in other pathosystems. Finally, we show that these classes of genes are under directional selection, and that this has predominantly targeted the Global Panzootic Lineage (BdGPL. Our analyses show that Bd manifests an unusually dynamic genome that may have been shaped by its association with the amphibian host. The rates of variation that we document likely explain the high levels of phenotypic variability that have been reported for Bd, and suggests that the dynamic genome of this pathogen has contributed to its success across multiple biomes and host-species.

  6. A novel single virus infection system reveals that influenza virus preferentially infects cells in g1 phase.

    Directory of Open Access Journals (Sweden)

    Ryuta Ueda

    Full Text Available BACKGROUND: Influenza virus attaches to sialic acid residues on the surface of host cells via the hemagglutinin (HA, a glycoprotein expressed on the viral envelope, and enters into the cytoplasm by receptor-mediated endocytosis. The viral genome is released and transported in to the nucleus, where transcription and replication take place. However, cellular factors affecting the influenza virus infection such as the cell cycle remain uncharacterized. METHODS/RESULTS: To resolve the influence of cell cycle on influenza virus infection, we performed a single-virus infection analysis using optical tweezers. Using this newly developed single-virus infection system, the fluorescence-labeled influenza virus was trapped on a microchip using a laser (1064 nm at 0.6 W, transported, and released onto individual H292 human lung epithelial cells. Interestingly, the influenza virus attached selectively to cells in the G1-phase. To clarify the molecular differences between cells in G1- and S/G2/M-phase, we performed several physical and chemical assays. Results indicated that: 1 the membranes of cells in G1-phase contained greater amounts of sialic acids (glycoproteins than the membranes of cells in S/G2/M-phase; 2 the membrane stiffness of cells in S/G2/M-phase is more rigid than those in G1-phase by measurement using optical tweezers; and 3 S/G2/M-phase cells contained higher content of Gb3, Gb4 and GlcCer than G1-phase cells by an assay for lipid composition. CONCLUSIONS: A novel single-virus infection system was developed to characterize the difference in influenza virus susceptibility between G1- and S/G2/M-phase cells. Differences in virus binding specificity were associated with alterations in the lipid composition, sialic acid content, and membrane stiffness. This single-virus infection system will be useful for studying the infection mechanisms of other viruses.

  7. Selection of mesenchymal-like metastatic cells in primary tumors – an in silico investigation

    Directory of Open Access Journals (Sweden)

    Vipin eNarang

    2012-04-01

    Full Text Available In order to metastasize, cancer cells must undergo phenotypic transition from an anchorage-dependent form to a motile form via a process referred to as epithelial to mesenchymal transition (EMT. It is currently unclear whether metastatic cells emerge late during tumor progression by successive accumulation of mutations, or whether they derive from distinct cell populations already present during the early stages of tumorigenesis. Similarly, the selective pressures that drive metastasis are poorly understood. Selection of cancer cells with increased proliferative capacity and enhanced survival characteristics may explain how some transformations promote a metastatic phenotype. However, it is difficult to explain how disseminated mesenchymal-like cancer cells can be subjected to such selective pressure, since these cells usually remain dormant for prolonged periods of time. In the current study, we have used in silico modeling and simulation to investigate the hypothesis that mesenchymal-like cancer cells evolve during the early stages of primary tumor development, and that these cells exhibit survival and proliferative advantages within the tumor microenvironment. In an agent-based tumor microenvironment model, cancer cell agents with distinct sets of attributes governing nutrient consumption, proliferation, apoptosis, random motility and cell adhesion were allowed to compete for space and nutrients. These simulation data indicated that mesenchymal-like cancer cells displaying high motility and low adhesion proliferate more rapidly and display a survival advantage over epithelial-like cancer cells. Furthermore, the presence of mesenchymal-like cells within the primary tumor influences the macroscopic properties, emergent morphology and growth rate of tumors.

  8. Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging

    OpenAIRE

    Hua, Wei; Sheff, David; Toomre, Derek; Mellman, Ira

    2006-01-01

    Although epithelial cells are known to exhibit a polarized distribution of membrane components, the pathways responsible for delivering membrane proteins to their appropriate domains remain unclear. Using an optimized approach to three-dimensional live cell imaging, we have visualized the transport of newly synthesized apical and basolateral membrane proteins in fully polarized filter-grown Madin–Darby canine kidney cells. We performed a detailed quantitative kinetic analysis of trans-Golgi n...

  9. Single-Cell RNA Sequencing Reveals T Helper Cells Synthesizing Steroids De Novo to Contribute to Immune Homeostasis

    Directory of Open Access Journals (Sweden)

    Bidesh Mahata

    2014-05-01

    Full Text Available T helper 2 (Th2 cells regulate helminth infections, allergic disorders, tumor immunity, and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2 signaling molecules. Although steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterized. Here, we demonstrate production of the steroid pregnenolone by Th2 cells in vitro and in vivo in a helminth infection model. Single-cell RNA sequencing and quantitative PCR analysis suggest that pregnenolone synthesis in Th2 cells is related to immunosuppression. In support of this, we show that pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching. We also show that steroidogenic Th2 cells inhibit Th cell proliferation in a Cyp11a1 enzyme-dependent manner. We propose pregnenolone as a “lymphosteroid,” a steroid produced by lymphocytes. We speculate that this de novo steroid production may be an intrinsic phenomenon of Th2-mediated immune responses to actively restore immune homeostasis.

  10. Single-cell RNA sequencing: revealing human pre-implantation development, pluripotency and germline development.

    Science.gov (United States)

    Petropoulos, S; Panula, S P; Schell, J P; Lanner, F

    2016-09-01

    Early human development is a dynamic, heterogeneous, complex and multidimensional process. During the first week, the single-cell zygote undergoes eight to nine rounds of cell division generating the multicellular blastocyst, which consists of hundreds of cells forming spatially organized embryonic and extra-embryonic tissues. At the level of transcription, degradation of maternal RNA commences at around the two-cell stage, coinciding with embryonic genome activation. Although numerous efforts have recently focused on delineating this process in humans, many questions still remain as thorough investigation has been limited by ethical issues, scarce availability of human embryos and the presence of minute amounts of DNA and RNA. In vitro cultures of embryonic stem cells provide some insight into early human development, but such studies have been confounded by analysis on a population level failing to appreciate cellular heterogeneity. Recent technical developments in single-cell RNA sequencing have provided a novel and powerful tool to explore the early human embryo in a systematic manner. In this review, we will discuss the advantages and disadvantages of the techniques utilized to specifically investigate human development and consider how the technology has yielded new insights into pre-implantation development, embryonic stem cells and the establishment of the germ line. PMID:27046137

  11. Solid-State NMR on bacterial cells: selective cell wall signal enhancement and resolution improvement using dynamic nuclear polarization

    International Nuclear Information System (INIS)

    Dynamic nuclear polarization (DNP) enhanced solid-state nuclear magnetic resonance (NMR) has recently emerged as a powerful technique for the study of material surfaces. In this study, we demonstrate its potential to investigate cell surface in intact cells. Using Bacillus subtilis bacterial cells as an example, it is shown that the polarizing agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL) has a strong binding affinity to cell wall polymers (peptidoglycan). This particular interaction is thoroughly investigated with a systematic study on extracted cell wall materials, disrupted cells, and entire cells, which proved that TOTAPOL is mainly accumulating in the cell wall. This property is used on one hand to selectively enhance or suppress cell wall signals by controlling radical concentrations and on the other hand to improve spectral resolution by means of a difference spectrum. Comparing DNP-enhanced and conventional solid-state NMR, an absolute sensitivity ratio of 24 was obtained on the entire cell sample. This important increase in sensitivity together with the possibility of enhancing specifically cell wall signals and improving resolution really opens new avenues for the use of DNP-enhanced solid-state NMR as an on-cell investigation tool. (authors)

  12. A systematic analysis of cell cycle regulators in yeast reveals that most factors act independently of cell size to control initiation of division.

    Directory of Open Access Journals (Sweden)

    Scott A Hoose

    Full Text Available Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms.

  13. Characterization of a null allelic mutant of the rice NAL1 gene reveals its role in regulating cell division.

    Directory of Open Access Journals (Sweden)

    Dan Jiang

    Full Text Available Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1 show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1, nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogeneous expression of NAL1 in fission yeast (Schizosaccharomyces pombe further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division.

  14. Systems Analyses Reveal Shared and Diverse Attributes of Oct4 Regulation in Pluripotent Cells

    DEFF Research Database (Denmark)

    Ding, Li; Paszkowski-Rogacz, Maciej; Winzi, Maria;

    2015-01-01

    Oct4, a key regulator of pluripotency. Our data signify that there are similarities, but also fundamental differences in Oct4 regulation in EpiSCs versus embryonic stem cells (ESCs). Through multiparametric data analyses, we predict that Tox4 is associating with the Paf1C complex, which maintains cell...... identity in both cell types, and validate that this protein-protein interaction exists in ESCs and EpiSCs. We also identify numerous knockdowns that increase Oct4 expression in EpiSCs, indicating that, in stark contrast to ESCs, Oct4 is under active repressive control in EpiSCs. These studies provide a...

  15. Diversifying selection in the wheat stem rust fungus acts predominantly on pathogen-associated gene families and reveals candidate effectors.

    Science.gov (United States)

    Sperschneider, Jana; Ying, Hua; Dodds, Peter N; Gardiner, Donald M; Upadhyaya, Narayana M; Singh, Karam B; Manners, John M; Taylor, Jennifer M

    2014-01-01

    Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus, Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with target host defense proteins. Previous efforts to understand virulence largely relied on the prediction of secreted, small and cysteine-rich proteins as candidate effectors and thus delivered an overwhelming number of candidates. Here, we implement an alternative analysis strategy that uses the signal of adaptive evolution as a line of evidence for effector function, combined with comparative information and expression data. We demonstrate that in planta up-regulated genes that are rapidly evolving are found almost exclusively in pathogen-associated gene families, affirming the impact of host-pathogen co-evolution on genome structure and the adaptive diversification of specialized gene families. In particular, we predict 42 effector candidates that are conserved only across pathogens, induced during infection and rapidly evolving. One of our top candidates has recently been shown to induce genotype-specific hypersensitive cell death in wheat. This shows that comparative genomics incorporating the evolutionary signal of adaptation is powerful for predicting effector candidates for laboratory verification. Our system can be applied to a wide range of pathogens and will give insight into host-pathogen dynamics, ultimately leading to progress in strategies for disease control. PMID:25225496

  16. The selective role of ECM components on cell adhesion, morphology, proliferation and communication in vitro

    International Nuclear Information System (INIS)

    Cell binding to the extracellular matrix (ECM) is essential for cell and tissue functions. In this context, each tissue consists of a unique ECM composition, which may be responsible for tissue-specific cell responses. Due to the complexity of ECM-cell interactions—which depend on the interplay of inside-out and outside-in signaling cascades, cell and tissue specificity of ECM-guidance is poorly understood. In this paper, we investigate the role of different ECM components like laminin, fibronectin, and collagen type I with respect to the essential cell behaviour patterns: attachment dynamics such as adhesion kinetic and force, formation of focal adhesion complexes, morphology, proliferation, and intercellular communication. A detailed in vitro comparison of fibroblasts, endothelial cells, osteoblasts, smooth muscle cells, and chondrocytes reveals significant differences in their cell responses to the ECM: cell behaviour follows a cell specific ligand priority ranking, which was independent of the cell type origin. Fibroblasts responded best to fibronectin, chondrocytes best to collagen I, the other cell types best to laminin. This knowledge is essential for optimization of tissue-biomaterial interfaces in all tissue engineering applications and gives insight into tissue-specific cell guidance. -- Highlights: • We analyse the impact of ECM components on cell behaviour in vitro. • We compare five different cell types, using the same culture conditions. • The ECM significantly guides all cell responses. • Cell behaviour follows a cell specific ligand-priority ranking. • This gives insight in tissue formation and is essential for biomedical applications

  17. Proteomic Profiling of Iron Overload-Induced Human Hepatic Cells Reveals Activation of TLR2-Mediated Inflammatory Response

    Directory of Open Access Journals (Sweden)

    Xiang Li

    2016-03-01

    Full Text Available Background: Hepatic iron overload is common in patients who have undergone hematopoietic cell transplantation (HCT and may predispose to peri- and post-HCT toxicity. To better reveal more molecules that might be involved in iron overload-induced liver injury, we utilized proteomics to investigate differentially expressed proteins in iron overload-induced hepatocytes vs. untreated hepatocytes. Methods and Results: HH4 hepatocytes were exposed to ferric ammonium citrate (FAC to establish an in vitro iron overload model. Differentially expressed proteins initiated by the iron overload were studied by two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS analysis. We identified 93 proteins whose quantity statistically significantly changes under excess hepatocyte iron conditions. Gene Ontology (GO analysis showed that these differentially expressed proteins in HH4 cells are involved in various biological process including endocytosis, response to wounding, di-, trivalent inorganic cation homeostasis, inflammatory response, positive regulation of cytokine production, and etc. Meanwhile, proteomics data revealed protein level of TLR2 and IL6ST significantly increased 7 times and 2.9 times, respectively, in iron overloaded HH4 cells. Our subsequent experiments detected that FAC-treated HH4 cells can activate IL6 expression through TLR2-mediated inflammatory responses via the NF-κB pathway. Conclusions: In this study, we demonstrated that iron overload induced hepatocytes triggering TLR2-mediated inflammatory response via NF-κB signaling pathway in HH4 cells.

  18. Embryonic stem cell-like features of testicular carcinoma in situ revealed by genome-wide gene expression profiling

    DEFF Research Database (Denmark)

    Almstrup, Kristian; Hoei-Hansen, Christina E; Wirkner, Ute;

    2004-01-01

    their stoichiometry on progression into embryonic carcinoma. We compared the CIS expression profile with patterns reported in embryonic stem cells (ESCs), which revealed a substantial overlap that may be as high as 50%. We also demonstrated an over-representation of expressed genes in regions of 17q and......Carcinoma in situ (CIS) is the common precursor of histologically heterogeneous testicular germ cell tumors (TGCTs), which in recent decades have markedly increased and now are the most common malignancy of young men. Using genome-wide gene expression profiling, we identified >200 genes highly...

  19. Whole exome sequencing reveals recurrent mutations in BRCA2 and FAT genes in acinar cell carcinomas of the pancreas

    OpenAIRE

    Toru Furukawa; Hitomi Sakamoto; Shoko Takeuchi; Mitra Ameri; Yuko Kuboki; Toshiyuki Yamamoto; Takashi Hatori; Masakazu Yamamoto; Masanori Sugiyama; Nobuyuki Ohike; Hiroshi Yamaguchi; Michio Shimizu; Noriyuki Shibata; Kyoko Shimizu; Keiko Shiratori

    2015-01-01

    Acinar cell carcinoma of the pancreas is a rare tumor with a poor prognosis. Compared to pancreatic ductal adenocarcinoma, its molecular features are poorly known. We studied a total of 11 acinar cell carcinomas, including 3 by exome and 4 by target sequencing. Exome sequencing revealed 65 nonsynonymous mutations and 22 indels with a mutation rate of 3.4 mutations/Mb per tumor, on average. By accounting for not only somatic but also germline mutations with loss of the wild-type allele, we ide...

  20. Genomic analysis reveals a potential role for cell cycle perturbation in HCV-mediated apoptosis of cultured hepatocytes.

    Directory of Open Access Journals (Sweden)

    Kathie-Anne Walters

    2009-01-01

    Full Text Available The mechanisms of liver injury associated with chronic HCV infection, as well as the individual roles of both viral and host factors, are not clearly defined. However, it is becoming increasingly clear that direct cytopathic effects, in addition to immune-mediated processes, play an important role in liver injury. Gene expression profiling during multiple time-points of acute HCV infection of cultured Huh-7.5 cells was performed to gain insight into the cellular mechanism of HCV-associated cytopathic effect. Maximal induction of cell-death-related genes and appearance of activated caspase-3 in HCV-infected cells coincided with peak viral replication, suggesting a link between viral load and apoptosis. Gene ontology analysis revealed that many of the cell-death genes function to induce apoptosis in response to cell cycle arrest. Labeling of dividing cells in culture followed by flow cytometry also demonstrated the presence of significantly fewer cells in S-phase in HCV-infected relative to mock cultures, suggesting HCV infection is associated with delayed cell cycle progression. Regulation of numerous genes involved in anti-oxidative stress response and TGF-beta1 signaling suggest these as possible causes of delayed cell cycle progression. Significantly, a subset of cell-death genes regulated during in vitro HCV infection was similarly regulated specifically in liver tissue from a cohort of HCV-infected liver transplant patients with rapidly progressive fibrosis. Collectively, these data suggest that HCV mediates direct cytopathic effects through deregulation of the cell cycle and that this process may contribute to liver disease progression. This in vitro system could be utilized to further define the cellular mechanism of this perturbation.

  1. BRCA1/2 mutation analysis in 41 ovarian cell lines reveals only one functionally deleterious BRCA1 mutation.

    LENUS (Irish Health Repository)

    Stordal, Britta

    2013-06-01

    Mutations in BRCA1\\/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1\\/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1\\/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1\\/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors. The BRCA1\\/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1\\/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1\\/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1\\/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311. The incidence of BRCA1\\/2 deleterious mutations 1\\/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective

  2. Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching.

    Science.gov (United States)

    Horns, Felix; Vollmers, Christopher; Croote, Derek; Mackey, Sally F; Swan, Gary E; Dekker, Cornelia L; Davis, Mark M; Quake, Stephen R

    2016-01-01

    Antibody class switching is a feature of the adaptive immune system which enables diversification of the effector properties of antibodies. Even though class switching is essential for mounting a protective response to pathogens, the in vivo patterns and lineage characteristics of antibody class switching have remained uncharacterized in living humans. Here we comprehensively measured the landscape of antibody class switching in human adult twins using antibody repertoire sequencing. The map identifies how antibodies of every class are created and delineates a two-tiered hierarchy of class switch pathways. Using somatic hypermutations as a molecular clock, we discovered that closely related B cells often switch to the same class, but lose coherence as somatic mutations accumulate. Such correlations between closely related cells exist when purified B cells class switch in vitro, suggesting that class switch recombination is directed toward specific isotypes by a cell-autonomous imprinted state. PMID:27481325

  3. Comparisons of microRNA patterns in plasma before and after tumor removal reveal new biomarkers of lung squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Vasily N Aushev

    Full Text Available Lung cancer is the major human malignancy, accounting for 30% of all cancer-related deaths worldwide. Poor survival of lung cancer patients, together with late diagnosis and resistance to classic chemotherapy, highlights the need for identification of new biomarkers for early detection. Among different cancer biomarkers, small non-coding RNAs called microRNAs (miRNAs are considered the most promising, owing to their remarkable stability, their cancer-type specificity, and their presence in body fluids. However, results of multiple previous attempts to identify circulating miRNAs specific for lung cancer are inconsistent, likely due to two main reasons: prominent variability in blood miRNA content among individuals and difficulties in distinguishing tumor-relevant miRNAs in the blood from their non-tumor counterparts. To overcome these impediments, we compared circulating miRNA profiles in patients with lung squamous cell carcinoma (SCC before and after tumor removal, assuming that the levels of all tumor-relevant miRNAs would drop after the surgery. Our results revealed a specific panel of the miRNAs (miR-205, -19a, -19b, -30b, and -20a whose levels decreased strikingly in the blood of patients after lung SCC surgery. Interestingly, miRNA profiling of plasma fractions of lung SCC patients revealed high levels of these miRNA species in tumor-specific exosomes; additionally, some of these miRNAs were also found to be selectively secreted to the medium by cultivated lung cancer cells. These results strengthen the notion that tumor cells secrete miRNA-containing exosomes into circulation, and that miRNA profiling of the exosomal plasma fraction may reveal powerful cancer biomarkers.

  4. Histopathologic characterization of the BTBR mouse model of autistic-like behavior reveals selective changes in neurodevelopmental proteins and adult hippocampal neurogenesis

    Directory of Open Access Journals (Sweden)

    Stephenson Diane T

    2011-05-01

    doublecortin, PSA-NCAM and NeuroD immunoreactive cells in the subgranular zone of the dentate gyrus, and a marked reduction in the number of 5-bromo-2'-deoxyuridine (BrdU positive progenitors. Furthermore, a significant and profound reduction in BDNF mRNA was seen in the BTBR dentate gyrus. No significant differences were seen in the expression of AchE, mossy fiber synapses or immunoreactivities of microtubule-associated protein MAP2, parvalbumin and glutamate decarboxylase GAD65 or GAD67 isoforms. Conclusions We documented modest and selective alterations in glia, neurons and synapses in BTBR forebrain, along with reduced neurogenesis in the adult hippocampus. Of all markers examined, the most distinctive changes were seen in the neurodevelopmental proteins NG2, PSA-NCAM, NeuroD and DCX. Our results are consistent with aberrant development of the nervous system in BTBR mice, and may reveal novel substrates to link callosal abnormalities and autistic behaviors. The changes that we observed in the BTBR mice suggest potential novel therapeutic strategies for intervention in autism spectrum disorders.

  5. Id4 knockdown during zebrafish development revealed its functional role in neural stem cell survival

    OpenAIRE

    Patlola, Santosh

    2010-01-01

    Id4 (Inhibitor of DNA binding 4 / Inhibitor of Differentiation 4) is one of the four members of Id protein family that antagonise the function of basic helix-loop-helix (bHLH) transcriptional regulators. In the mouse it has been shown that Id4 plays an important role in the timing of neural stem and progenitor cell differentiation and knockout mice exhibited premature neural stem cell differentiation resulting in significantly smaller brains. To further establish the molecular mechanism under...

  6. Dynamics between Cancer Cell Subpopulations Reveals a Model Coordinating with Both Hierarchical and Stochastic Concepts

    OpenAIRE

    Wang, Weikang; Quan, Yi; Fu, Qibin; Liu, Yu; Liang, Ying; Wu, Jingwen; Yang, Gen; Luo, Chunxiong; Ouyang, Qi; Wang, Yugang

    2014-01-01

    Tumors are often heterogeneous in which tumor cells of different phenotypes have distinct properties. For scientific and clinical interests, it is of fundamental importance to understand their properties and the dynamic variations among different phenotypes, specifically under radio- and/or chemo-therapy. Currently there are two controversial models describing tumor heterogeneity, the cancer stem cell (CSC) model and the stochastic model. To clarify the controversy, we measured probabilities ...

  7. Atomic force microscope elastography reveals phenotypic differences in alveolar cell stiffness

    OpenAIRE

    Azeloglu, Evren U.; Bhattacharya, Jahar; COSTA, KEVIN D.

    2008-01-01

    To understand the connection between alveolar mechanics and key biochemical events such as surfactant secretion, one first needs to characterize the underlying mechanical properties of the lung parenchyma and its cellular constituents. In this study, the mechanics of three major cell types from the neonatal rat lung were studied; primary alveolar type I (AT1) and type II (AT2) epithelial cells and lung fibroblasts were isolated using enzymatic digestion. Atomic force microscopy indentation wa...

  8. Combined In Silico and In Vivo Analyses Reveal Role of Hes1 in Taste Cell Differentiation

    OpenAIRE

    Ota, Masato S.; Yoshiyuki Kaneko; Kaori Kondo; Soichi Ogishima; Hiroshi Tanaka; Kazuhiro Eto; Takashi Kondo

    2009-01-01

    Author Summary The sensation of taste is composed of five basic modalities: sweet, bitter, umami, sour, and salty. Specialized taste cells perceive the various chemical cues within food. About 100 taste cells assemble into onion-shaped clusters called taste buds, which are located on taste papillae in the tongue epithelium and on oral mucosa. Of the five taste modalities, the taste stimulants responsible for sweet, bitter, and umami tastes are recognized by a group of G protein–coupled taste ...

  9. Engineering Multi-Walled Carbon Nanotube Therapeutic Bionanofluids to Selectively Target Papillary Thyroid Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Idit Dotan

    Full Text Available The incidence of papillary thyroid carcinoma (PTC has risen steadily over the past few decades as well as the recurrence rates. It has been proposed that targeted ablative physical therapy could be a therapeutic modality in thyroid cancer. Targeted bio-affinity functionalized multi-walled carbon nanotubes (BioNanofluid act locally, to efficiently convert external light energy to heat thereby specifically killing cancer cells. This may represent a promising new cancer therapeutic modality, advancing beyond conventional laser ablation and other nanoparticle approaches.Thyroid Stimulating Hormone Receptor (TSHR was selected as a target for PTC cells, due to its wide expression. Either TSHR antibodies or Thyrogen or purified TSH (Thyrotropin were chemically conjugated to our functionalized Bionanofluid. A diode laser system (532 nm was used to illuminate a PTC cell line for set exposure times. Cell death was assessed using Trypan Blue staining.TSHR-targeted BioNanofluids were capable of selectively ablating BCPAP, a TSHR-positive PTC cell line, while not TSHR-null NSC-34 cells. We determined that a 2:1 BCPAP cell:α-TSHR-BioNanofluid conjugate ratio and a 30 second laser exposure killed approximately 60% of the BCPAP cells, while 65% and >70% of cells were ablated using Thyrotropin- and Thyrogen-BioNanofluid conjugates, respectively. Furthermore, minimal non-targeted killing was observed using selective controls.A BioNanofluid platform offering a potential therapeutic path for papillary thyroid cancer has been investigated, with our in vitro results suggesting the development of a potent and rapid method of selective cancer cell killing. Therefore, BioNanofluid treatment emphasizes the need for new technology to treat patients with local recurrence and metastatic disease who are currently undergoing either re-operative neck explorations, repeated administration of radioactive iodine and as a last resort external beam radiation or chemotherapy, with

  10. Engineering Multi-Walled Carbon Nanotube Therapeutic Bionanofluids to Selectively Target Papillary Thyroid Cancer Cells

    Science.gov (United States)

    Paliouras, Miltiadis; Mitmaker, Elliot J.; Trifiro, Mark A.

    2016-01-01

    Background The incidence of papillary thyroid carcinoma (PTC) has risen steadily over the past few decades as well as the recurrence rates. It has been proposed that targeted ablative physical therapy could be a therapeutic modality in thyroid cancer. Targeted bio-affinity functionalized multi-walled carbon nanotubes (BioNanofluid) act locally, to efficiently convert external light energy to heat thereby specifically killing cancer cells. This may represent a promising new cancer therapeutic modality, advancing beyond conventional laser ablation and other nanoparticle approaches. Methods Thyroid Stimulating Hormone Receptor (TSHR) was selected as a target for PTC cells, due to its wide expression. Either TSHR antibodies or Thyrogen or purified TSH (Thyrotropin) were chemically conjugated to our functionalized Bionanofluid. A diode laser system (532 nm) was used to illuminate a PTC cell line for set exposure times. Cell death was assessed using Trypan Blue staining. Results TSHR-targeted BioNanofluids were capable of selectively ablating BCPAP, a TSHR-positive PTC cell line, while not TSHR-null NSC-34 cells. We determined that a 2:1 BCPAP cell:α-TSHR-BioNanofluid conjugate ratio and a 30 second laser exposure killed approximately 60% of the BCPAP cells, while 65% and >70% of cells were ablated using Thyrotropin- and Thyrogen-BioNanofluid conjugates, respectively. Furthermore, minimal non-targeted killing was observed using selective controls. Conclusion A BioNanofluid platform offering a potential therapeutic path for papillary thyroid cancer has been investigated, with our in vitro results suggesting the development of a potent and rapid method of selective cancer cell killing. Therefore, BioNanofluid treatment emphasizes the need for new technology to treat patients with local recurrence and metastatic disease who are currently undergoing either re-operative neck explorations, repeated administration of radioactive iodine and as a last resort external beam

  11. Cellular Metabolism and Dose Reveal Carnitine-Dependent and -Independent Mechanisms of Butyrate Oxidation in Colorectal Cancer Cells.

    Science.gov (United States)

    Han, Anna; Bennett, Natalie; MacDonald, Amber; Johnstone, Megan; Whelan, Jay; Donohoe, Dallas R

    2016-08-01

    Dietary fiber has been suggested to suppress colorectal cancer development, although the mechanisms contributing to this beneficial effect remain elusive. Butyrate, a fermentation product of fiber, has been shown to have anti-proliferative and pro-apoptotic effects on colorectal cancer cells. The metabolic fate of butyrate in the cell is important in determining whether, it acts as an HDAC inhibitor or is consumed as a short-chain fatty acid. Non-cancerous colonocytes utilize butyrate as the primary energy source whereas cancerous colonocytes increase glucose utilization through the Warburg effect. In this study, we show that butyrate oxidation is decreased in cancerous colonocytes compared to non-cancerous colonocytes. We demonstrate that colorectal cancer cells utilize both a carnitine-dependent and carnitine-independent mechanism that contributes to butyrate oxidation. The carnitine-dependent mechanism is contingent on butyrate concentration. Knockdown of CPT1A in colorectal cancer cells abolishes butyrate oxidation. In terms of selectivity, the carnitine-dependent mechanism only regulated butyrate oxidation, as acetate and propionate oxidation were carnitine-independent. Carnitine decreased the action of butyrate as an HDAC inhibitor and suppressed induction of H3 acetylation by butyrate in colorectal cancer cells. Thus, diminished oxidation of butyrate is associated with decreased HDAC inhibition and histone acetylation. In relation to the mechanism, we find that dichloroacetate, which decreases phosphorylation of pyruvate dehydrogenase, increased butyrate oxidation and that this effect was carnitine-dependent. In conclusion, these data suggest that colorectal cancer cells decrease butyrate oxidation through inhibition of pyruvate dehydrogenase, which is carnitine-dependent, and provide insight into why butyrate shows selective effects toward colorectal cancer cells. J. Cell. Physiol. 231: 1804-1813, 2016. © 2015 Wiley Periodicals, Inc. PMID:26661480

  12. Melanoma chemotherapy leads to the selection of ABCB5-expressing cells.

    Directory of Open Access Journals (Sweden)

    Marine Chartrain

    Full Text Available Metastatic melanoma is the most aggressive skin cancer. Recently, phenotypically distinct subpopulations of tumor cells were identified. Among them, ABCB5-expressing cells were proposed to display an enhanced tumorigenicity with stem cell-like properties. In addition, ABCB5(+ cells are thought to participate to chemoresistance through a potential efflux function of ABCB5. Nevertheless, the fate of these cells upon drugs that are used in melanoma chemotherapy remains to be clarified. Here we explored the effect of anti-melanoma treatments on the ABCB5-expressing cells. Using a melanoma xenograft model (WM266-4, we observed in vivo that ABCB5-expressing cells are enriched after a temozolomide treatment that induces a significant tumor regression. These results were further confirmed in a preliminary study conducted on clinical samples from patients that received dacarbazine. In vitro, we showed that ABCB5-expressing cells selectively survive when exposed to dacarbazine, the reference treatment of metastatic melanoma, but also to vemurafenib, a new inhibitor of the mutated kinase V600E BRAF and other various chemotherapeutic drugs. Our results show that anti-melanoma chemotherapy might participate to the chemoresistance acquisition by selecting tumor cell subpopulations expressing ABCB5. This is of particular importance in understanding the relapses observed after anti-melanoma treatments and reinforces the interest of ABCB5 and ABCB5-expressing cells as potential therapeutic targets in melanoma.

  13. Selective photothermal efficiency of citrate capped gold nanoparticles for destruction of cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Raji, V. [Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram 695 581, Kerala (India); Kumar, Jatish [Division of photochemistry and photobiology, National Institute for Interdisciplinary Sciences and Technology (CSIR), Thiruvananthapuram 695 019, Kerala (India); Rejiya, C.S.; Vibin, M.; Shenoi, Vinesh N. [Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram 695 581, Kerala (India); Abraham, Annie, E-mail: annieab2@yahoo.co.in [Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram 695 581, Kerala (India)

    2011-08-15

    Gold nanoparticles are recently having much attention because of their increased applications in biomedical fields. In this paper, we demonstrated the photothermal efficacy of citrate capped gold nanoparticles (AuNPs) for the destruction of A431 cancer cells. Citrate capped AuNPs were synthesized successfully and characterized by UV-visible-NIR spectrophotometry and High Resolution Transmission Electron Microscopy (HR-TEM). Further, AuNPs were conjugated with epidermal growth factor receptor antibody (anti-EGFR) and applied for the selective photothermal therapy (PTT) of human epithelial cancer cells, A431. PTT experiments were conducted in four groups, Group I-control cells, Group II-cells treated with laser light alone, Group III-cells treated with unconjugated AuNP and further laser irradiation and Group IV-anti-EGFR conjugated AuNP treated cells irradiated by laser light. After laser irradiation, cell morphology changes that were examined using phase contrast microscopy along with the relevant biochemical parameters like lactate dehydrogenase activity, reactive oxygen species generation and caspase-3 activity were studied for all the groups to determine whether cell death occurs due to necrosis or apoptosis. From these results we concluded that, these immunotargeted nanoparticles could selectively induce cell death via ROS mediated apoptosis when cells were exposed to a low power laser light.

  14. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion.

    Science.gov (United States)

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2015-01-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2-4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2-4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:26042727

  15. Plasma membrane microorganization of LR73 multidrug-resistant cells revealed by FCS

    Science.gov (United States)

    Winckler, Pascale; Jaffiol, Rodolphe; Cailler, Aurélie; Morjani, Hamid; Jeannesson, Pierre; Deturche, Régis

    2011-03-01

    Tumoral cells could present a multidrug resistance (MDR) to chemotherapeutic treatments. This drug resistance would be associated to biomechanisms occurring at the plasma membrane level, involving modification of membrane fluidity, drug permeability, presence of microdomains (rafts, caveolae...), and membrane proteins overexpression such as Pglycoprotein. Fluorescence correlation spectroscopy (FCS) is the relevant method to investigate locally the fluidity of biological membranes through the lateral diffusion of a fluorescent membrane probe. Thus, we use FCS to monitor the plasma membrane local organization of LR73 carcinoma cells and three derived multidrug-resistant cancer cells lines. Measurements were conducted at the single cell level, which enabled us to get a detailed overview of the plasma membrane microviscosity distribution of each cell line studied. Moreover, we propose 2D diffusion simulation based on a Monte Carlo model to investigate the membrane organisation in terms of microdomains. This simulation allows us to relate the differences in the fluidity distributions with microorganization changes in plasma membrane of MDR cells.

  16. Positive and negative selection of T cells in T-cell receptor transgenic mice expressing a bcl-2 transgene.

    OpenAIRE

    Strasser, A.; Harris, A W; von Boehmer, H; Cory, S

    1994-01-01

    To explore the role of bcl-2 in T-cell development, a bcl-2 transgene was introduced into mice expressing a T-cell receptor (TCR) transgene encoding reactivity for the mouse male antigen HY presented by the H-2Db class I antigen of the major histocompatibility complex (MHC). Normal thymic development is contingent on the ability of immature thymocytes to interact with self-MHC molecules presented by thymic stroma (positive selection). Thus, thymocyte numbers are low in femal...

  17. Cellular Architecture of Treponema pallidum: Novel Flagellum, Periplasmic Cone, and Cell Envelope as Revealed by Cryo-Electron Tomography

    OpenAIRE

    Liu, Jun; Howell, Jerrilyn K.; Bradley, Sherille D.; Zheng, Yesha; Zhou, Z. Hong; Norris, Steven J

    2010-01-01

    High resolution cryo-electron tomography (cryo-ET) was utilized to visualize Treponema pallidum, the causative agent of syphilis, at the molecular level. Three-dimensional (3-D) reconstructions from 304 infectious organisms revealed unprecedented cellular structures of this unusual member in the spirochetal family. High resolution cryo-ET reconstructions provided the detailed structures of the cell envelope, which is significantly different from that of gram-negative bacteria. The 4 nm lipid ...

  18. Selective release of microRNA species from normal and malignant mammary epithelial cells.

    Directory of Open Access Journals (Sweden)

    Lucy Pigati

    Full Text Available MicroRNAs (miRNAs in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.

  19. Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos

    Directory of Open Access Journals (Sweden)

    Hartung Odelya

    2007-12-01

    Full Text Available Abstract Background The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. Results We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression. Conclusion This paper describes the first example of multiplex RT-LATE-PCR and its utility, when

  20. Viral Transmission Dynamics at Single-Cell Resolution Reveal Transiently Immune Subpopulations Caused by a Carrier State Association

    Science.gov (United States)

    Cenens, William; Makumi, Angela; Govers, Sander K.; Lavigne, Rob; Aertsen, Abram

    2015-01-01

    Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22) throughout a population of its host (Salmonella Typhimurium) at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization. More specifically, an infection event ultimately leading to a lysogen first yielded a phage carrier cell harboring a polarly tethered P22 episome. Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free. However, since the phage carrier cell was shown to overproduce immunity factors that are cytoplasmically inherited by the P22-free daughter cell and further passed down to its siblings, a transiently resistant subpopulation was generated that upon dilution of these immunity factors again became susceptible to P22 infection. The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host. PMID:26720743

  1. An excitatory amacrine cell detects object motion and provides feature-selective input to ganglion cells in the mouse retina

    Science.gov (United States)

    Kim, Tahnbee; Soto, Florentina; Kerschensteiner, Daniel

    2015-01-01

    Retinal circuits detect salient features of the visual world and report them to the brain through spike trains of retinal ganglion cells. The most abundant ganglion cell type in mice, the so-called W3 ganglion cell, selectively responds to movements of small objects. Where and how object motion sensitivity arises in the retina is incompletely understood. In this study, we use 2-photon-guided patch-clamp recordings to characterize responses of vesicular glutamate transporter 3 (VGluT3)-expressing amacrine cells (ACs) to a broad set of visual stimuli. We find that these ACs are object motion sensitive and analyze the synaptic mechanisms underlying this computation. Anatomical circuit reconstructions suggest that VGluT3-expressing ACs form glutamatergic synapses with W3 ganglion cells, and targeted recordings show that the tuning of W3 ganglion cells' excitatory input matches that of VGluT3-expressing ACs' responses. Synaptic excitation of W3 ganglion cells is diminished, and responses to object motion are suppressed in mice lacking VGluT3. Object motion, thus, is first detected by VGluT3-expressing ACs, which provide feature-selective excitatory input to W3 ganglion cells. DOI: http://dx.doi.org/10.7554/eLife.08025.001 PMID:25988808

  2. Selective cytotoxicity of marine algae extracts to several human leukemic cell lines

    OpenAIRE

    Harada, Hideki; Kamei, Yuto

    1997-01-01

    Extracts from 8 species of marine algae which showed selective cytotoxicity in our previous screening program, were further examined for cytotoxic spectra to five human leukemic cell lines. The extract from a red alga, Amphiroa zonata exhibited strong cytotoxicity to all human leukemic cell lines tested and murine leukemic cells L1210 at the final concentrations from 15 to 375 µg ml−1. Then the cytotoxicity was not found in normal human fibroblast HDF and murine normal cells NIH-3T3. The acti...

  3. Resistance of tumor cells to hydrogen peroxide as a factor of selection of highly metastatic cell variants in vivo

    International Nuclear Information System (INIS)

    The authors investigate the theory that tumor cells which catabolize H2O2 more actively may possibly have selected advantages in vivo (of different origin). The authors tested the sensitivity to H2O2 of parental cells of strain STHE (which did not progress in vivo) and 17 of its daughter variants isolated from lung tissue of experimental animals at different stages of formation of metastases (before and after their formation) and differing in metastatic activity. Intact cells were placed into test tube No. 6 of each series. After 30 min of exposure at 200C, cells treated with H2O2 and intact cells were washed out by centrifugation, and resuspended in nutritive medium containing 10% bovine serum and 3H-thymidine, and each sample was diffused at a volume of 2.0 ml of cell suspension in each of three scintillation vials. The proliferative pool of cells in each sample was determined according to incorporation (for 22 h at 370C) of 3H-thymidine into cell nuclei. Data concerning incorporation of 3H-thymidine was expressed for each sample of cells in percentages in relation to corresponding intact control (incorporation of label in a control culture of intact cells was taken as 100%). Each cell variant was investigated repeatedly in two-three more experiments

  4. Direct Measurement of Water States in Cryopreserved Cells Reveals Tolerance toward Ice Crystallization.

    Science.gov (United States)

    Huebinger, Jan; Han, Hong-Mei; Hofnagel, Oliver; Vetter, Ingrid R; Bastiaens, Philippe I H; Grabenbauer, Markus

    2016-02-23

    Complex living systems such as mammalian cells can be arrested in a solid phase by ultrarapid cooling. This allows for precise observation of cellular structures as well as cryopreservation of cells. The state of water, the main constituent of biological samples, is crucial for the success of cryogenic applications. Water exhibits many different solid states. If it is cooled extremely rapidly, liquid water turns into amorphous ice, also called vitreous water, a glassy and amorphous solid. For cryo-preservation, the vitrification of cells is believed to be mandatory for cell survival after freezing. Intracellular ice crystallization is assumed to be lethal, but experimental data on the state of water during cryopreservation are lacking. To better understand the water conditions in cells subjected to freezing protocols, we chose to directly analyze their subcellular water states by cryo-electron microscopy and tomography, cryoelectron diffraction, and x-ray diffraction both in the cryofixed state and after warming to different temperatures. By correlating the survival rates of cells with their respective water states during cryopreservation, we found that survival is less dependent on ice-crystal formation than expected. Using high-resolution cryo-imaging, we were able to directly show that cells tolerate crystallization of extra- and intracellular water. However, if warming is too slow, many small ice crystals will recrystallize into fewer but bigger crystals, which is lethal. The applied cryoprotective agents determine which crystal size is tolerable. This suggests that cryoprotectants can act by inhibiting crystallization or recrystallization, but they also increase the tolerance toward ice-crystal growth. PMID:26541066

  5. Gene Regulatory Network Analysis Reveals Differences in Site-specific Cell Fate Determination in Mammalian Brain

    Directory of Open Access Journals (Sweden)

    Gokhan eErtaylan

    2014-12-01

    Full Text Available Neurogenesis - the generation of new neurons - is an ongoing process that persists in the adult mammalian brain of several species, including humans. In this work we analyze two discrete brain regions: the subventricular zone (SVZ lining the walls of the lateral ventricles; and the subgranular zone (SGZ of the dentate gyrus of the hippocampus in mice and shed light on the SVZ and SGZ specific neurogenesis. We propose a computational model that relies on the construction and analysis of region specific gene regulatory networks from the publicly available data on these two regions. Using this model a number of putative factors involved in neuronal stem cell (NSC identity and maintenance were identified. We also demonstrate potential gender and niche-derived differences based on cell surface and nuclear receptors via Ar, Hif1a and Nr3c1.We have also conducted cell fate determinant analysis for SVZ NSC populations to Olfactory Bulb interneurons and SGZ NSC populations to the granule cells of the Granular Cell Layer. We report thirty-one candidate cell fate determinant gene pairs, ready to be validated. We focus on Ar - Pax6 in SVZ and Sox2 - Ncor1 in SGZ. Both pairs are expressed and localized in the suggested anatomical structures as shown by in situ hybridization and found to physically interact.Finally, we conclude that there are fundamental differences between SGZ and SVZ neurogenesis. We argue that these regulatory mechanisms are linked to the observed differential neurogenic potential of these regions. The presence of nuclear and cell surface receptors in the region specific regulatory circuits indicate the significance of niche derived extracellular factors, hormones and region specific factors such as the oxygen sensitivity, dictating SGZ and SVZ specific neurogenesis.

  6. Cell Selection Game for Densely-Deployed Sensor and Mobile Devices In 5G Networks Integrating Heterogeneous Cells and the Internet of Things

    OpenAIRE

    Lusheng Wang; Yamei Wang; Zhizhong Ding; Xiumin Wang

    2015-01-01

    With the rapid development of wireless networking technologies, the Internet of Things and heterogeneous cellular networks (HCNs) tend to be integrated to form a promising wireless network paradigm for 5G. Hyper-dense sensor and mobile devices will be deployed under the coverage of heterogeneous cells, so that each of them could freely select any available cell covering it and compete for resource with others selecting the same cell, forming a cell selection (CS) game between these devices. S...

  7. Development of cell death-based method for the selectivity screening of caspase-1 inhibitors

    DEFF Research Database (Denmark)

    Chopra, Puneet; Gupta, Shashank; Dastidar, Sunanda G; Ray, Abhijit

    2009-01-01

    used for the selectivity screening of multiple caspases in a biologically relevant context in a single assay. In this study, we have developed an assay in which DNA fragmentation, a hallmark of apoptosis, of Jurkat cell line was examined post induction with etoposide in the presence or absence of...... belonging to caspase-1 family (1, 4 and 5) are not present in the Jurkat cells or might not be involved in the etoposide-induced DNA fragmentation. Since the inhibition of caspases 3, 8 and 9 is accompanied by the down regulation of the activity of a cascade of caspases (caspases 2, 6, 7, 9 and 10......Caspase-1 selective inhibitors are novel therapeutic agents for inflammatory diseases. Selectivity assays for caspases can be initiated with purified enzyme, making these assays very costly and time consuming. Therefore, there is a need to develop a fast and reliable cell-based assay, which can be...

  8. Analysis of selective reflection spectrum in cholesteric liquid crystal cells for solar-ray controller

    Science.gov (United States)

    Ogiwara, Akifumi; Kakiuchida, Hiroshi

    2015-09-01

    The cholesteric liquid crystal (CLC) cells are fabricated by varying the concentration of various chiral dopants and liquid crystal (LC) diacrylate monomers. The wavelength and bandwidth of selective reflection spectrum in CLC cells are measured by a spectroscopic technique. The variation of the selective reflection spectrum in CLC cells is investigated by doping the different kinds of liquid crystal (LC) diacrylate monomers which stabilize a helical twisting structure by photopolymerization. The effects of the selective reflection spectrum on the visible and infrared lights in spectral solar irradiance are explained by the performance for a solar-ray controller based on the spectral solar irradiance for air mass 1.5 and the standard luminous efficiency function for photopic vision.

  9. Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells

    DEFF Research Database (Denmark)

    Hattori, Naoko; Niwa, Tohru; Kimura, Kana;

    2013-01-01

    Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which....... Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell...

  10. Antimicrobial activity, improved cell selectivity and mode of action of short PMAP-36-derived peptides against bacteria and Candida.

    Science.gov (United States)

    Lyu, Yinfeng; Yang, Yang; Lyu, Xiting; Dong, Na; Shan, Anshan

    2016-01-01

    Antimicrobial peptides (AMPs) have recently attracted a great deal of attention as promising antibiotic candidates, but some obstacles such as toxicity and high synthesis cost must be addressed before developing them further. For developing short peptides with improved cell selectivity, we designed a series of modified PMAP-36 analogues. Antimicrobial assays showed that decreasing chain length in a certain range retained the high antimicrobial activity of the parental peptide and reduced hemolysis. The 18-mer peptide RI18 exhibited excellent antimicrobial activity against both bacteria and fungi, and its hemolytic activity was observably lower than PMAP-36 and melittin. The selectivity indexes of RI18 against bacteria and fungi were improved approximately 19-fold and 108-fold, respectively, compared to PMAP-36. In addition, serum did not affect the antibacterial activity of RI18 against E. coli but inhibited the antifungal efficiency against C. albicans. Flow cytometry and electron microscopy observation revealed that RI18 killed microbial cells primarily by damaging membrane integrity, leading to whole cell lysis. Taken together, these results suggest that RI18 has potential for further therapeutic research against frequently-encountered bacteria and fungi. Meanwhile, modification of AMPs is a promising strategy for developing novel antimicrobials to overcome drug-resistance. PMID:27251456

  11. Optical nanoscopy to reveal structural and functional properties of liver cells (Presentation Recording)

    Science.gov (United States)

    McCourt, Peter; Huser, Thomas R.; Sørensen, Karen K.; Øie, Cristina I.; Mönkemöller, Viola; Ahluwalia, Balpreet S.

    2015-08-01

    The advent of optical nanoscopy has provided an opportunity to study fundamental properties of nanoscale biological functions, such as liver sinusoidal endothelial cells (LSEC) and their fenestrations. The fenestrations are nano-pores (50-200 nm) on the LSEC plasma membrane that allow free passage of molecules through cells. The fenestrated LSEC also hase a voracious appetite for waste molecules, viruses and nanoparticles. LSEC daily remove huge amounts of waste, nanoparticles and virus from the blood. Pharmaceuticals also need to pass through these fenestrations to be activated (e.g. cholesterol reducing statins) or detoxified by hepatocytes. And, when we age, our LSEC fenestrations become smaller and fewer. Today, we study these cells and structures using either conventional light microscopy on living cells, or high-resolution (but static) methods such as transmission and scanning electron microscopy on fixed (i.e. dead) tissue. Such methods, while very powerful, yield no real time information about the uptake of virus or nanoparticles, nor any information about fenestration dynamics. Therefore, to study LS-SEC, we are now using optical nanoscopy methods, and developing our own, to map their functions in 4 dimensions. Attaining this goal will shed new light on the cell biology of the liver and how it keeps us alive. This paper describes the challenges of studying LS-SEC with light microscopy, as well as current and potential solutions to this challenge using optical nanoscopy.

  12. Tracking of plus-ends reveals microtubule functional diversity in different cell types

    Science.gov (United States)

    Shaebani, M. Reza; Pasula, Aravind; Ott, Albrecht; Santen, Ludger

    2016-07-01

    Many cellular processes are tightly connected to the dynamics of microtubules (MTs). While in neuronal axons MTs mainly regulate intracellular trafficking, they participate in cytoskeleton reorganization in many other eukaryotic cells, enabling the cell to efficiently adapt to changes in the environment. We show that the functional differences of MTs in different cell types and regions is reflected in the dynamic properties of MT tips. Using plus-end tracking proteins EB1 to monitor growing MT plus-ends, we show that MT dynamics and life cycle in axons of human neurons significantly differ from that of fibroblast cells. The density of plus-ends, as well as the rescue and catastrophe frequencies increase while the growth rate decreases toward the fibroblast cell margin. This results in a rather stable filamentous network structure and maintains the connection between nucleus and membrane. In contrast, plus-ends are uniformly distributed along the axons and exhibit diverse polymerization run times and spatially homogeneous rescue and catastrophe frequencies, leading to MT segments of various lengths. The probability distributions of the excursion length of polymerization and the MT length both follow nearly exponential tails, in agreement with the analytical predictions of a two-state model of MT dynamics.

  13. Actin marker lines in grapevine reveal a gatekeeper function of guard cells.

    Science.gov (United States)

    Guan, Xin; Buchholz, Günther; Nick, Peter

    2014-08-15

    Resistance to abiotic and biotic stress is a central topic for sustainable agriculture, especially in grapevine, one of the field crops with the highest economic output per acreage. As early cellular factors for plant defense, actin microfilaments (AF) are of high relevance. We therefore generated a transgenic actin marker line for grapevine by expressing a fusion protein between green fluorescent protein and the second actin-binding domain of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. Based on this first cytoskeletal-marker line in grapevine, the response of AFs to phytopathogenic microorganisms could be followed in vivo. Upon inoculation with fluorescently labeled strains of phytopathogenic bacteria, actin responses were confined to the guard cells. In contrast, upon contact with zoospores of Plasmopara viticola, not only the guard cells, but also epidermal pavement cells, where no zoospores had attached responded with the formation of a perinuclear actin basket. Our data support the hypothesis that guard cells act as pacemakers of defense, dominating the responses of the remaining epidermal cells. PMID:24973589

  14. Tracking of plus-ends reveals microtubule functional diversity in different cell types.

    Science.gov (United States)

    Shaebani, M Reza; Pasula, Aravind; Ott, Albrecht; Santen, Ludger

    2016-01-01

    Many cellular processes are tightly connected to the dynamics of microtubules (MTs). While in neuronal axons MTs mainly regulate intracellular trafficking, they participate in cytoskeleton reorganization in many other eukaryotic cells, enabling the cell to efficiently adapt to changes in the environment. We show that the functional differences of MTs in different cell types and regions is reflected in the dynamic properties of MT tips. Using plus-end tracking proteins EB1 to monitor growing MT plus-ends, we show that MT dynamics and life cycle in axons of human neurons significantly differ from that of fibroblast cells. The density of plus-ends, as well as the rescue and catastrophe frequencies increase while the growth rate decreases toward the fibroblast cell margin. This results in a rather stable filamentous network structure and maintains the connection between nucleus and membrane. In contrast, plus-ends are uniformly distributed along the axons and exhibit diverse polymerization run times and spatially homogeneous rescue and catastrophe frequencies, leading to MT segments of various lengths. The probability distributions of the excursion length of polymerization and the MT length both follow nearly exponential tails, in agreement with the analytical predictions of a two-state model of MT dynamics. PMID:27461361

  15. Fluorescent Nanocrystals Reveal Regulated Portals of Entry into and Between the Cells of Hydra

    Science.gov (United States)

    Tortiglione, Claudia; Quarta, Alessandra; Malvindi, Maria Ada; Tino, Angela; Pellegrino, Teresa

    2009-01-01

    Initially viewed as innovative carriers for biomedical applications, with unique photophysical properties and great versatility to be decorated at their surface with suitable molecules, nanoparticles can also play active roles in mediating biological effects, suggesting the need to deeply investigate the mechanisms underlying cell-nanoparticle interaction and to identify the molecular players. Here we show that the cell uptake of fluorescent CdSe/CdS quantum rods (QRs) by Hydra vulgaris, a simple model organism at the base of metazoan evolution, can be tuned by modifying nanoparticle surface charge. At acidic pH, amino-PEG coated QRs, showing positive surface charge, are actively internalized by tentacle and body ectodermal cells, while negatively charged nanoparticles are not uptaken. In order to identify the molecular factors underlying QR uptake at acidic pH, we provide functional evidence of annexins involvement and explain the QR uptake as the combined result of QR positive charge and annexin membrane insertion. Moreover, tracking QR labelled cells during development and regeneration allowed us to uncover novel intercellular trafficking and cell dynamics underlying the remarkable plasticity of this ancient organism. PMID:19888325

  16. Gene expression profiling reveals novel protective effects of Aminaphtone on ECV304 endothelial cells.

    Science.gov (United States)

    Salazar, Giulia; Bellocchi, Chiara; Todoerti, Katia; Saporiti, Federica; Piacentini, Luca; Scorza, Raffaella; Colombo, Gualtiero I

    2016-07-01

    Aminaphtone, a drug used in the treatment of chronic venous insufficiency (CVI), showed a remarkable role in the modulation of several vasoactive factors, like endothelin-1 and adhesion molecules. We analysed in vitro the effects of Aminaphtone on whole-genome gene expression and production of different inflammatory proteins. ECV-304 endothelial cells were stimulated with IL-1β 100U/ml in the presence or absence of Aminaphtone 6μg/ml. Gene expression profiles were compared at 1, 3, and 6h after stimulation by microarray. Supernatants of ECV-304 cultures were analysed at 3, 6, 12, and 24h by multiplex ELISA for production of several cytokine and chemokines. Microarrays showed a significant down-regulation at all times of a wide range of inflammatory genes. Aminaphtone appeared also able to modulate the regulation of immune response process (down-regulating cytokine biosynthesis, transcripts involved in lymphocyte differentiation and cell proliferation, and cytokine-cytokine receptor interaction) and to regulate genes engaged in homeostasis, secretion, body fluid levels, response to hypoxia, cell division, and cell-to-cell communication and signalling. Results were confirmed and extended analysing the secretome, which showed significant reduction of the release of 14 cytokines and chemokines. These effects are predicted to be mediated by interaction with different transcription factors. Aminaphtone was able to modulate the expression of inflammatory molecules relevant to the pathogenesis of several conditions in which the endothelial dysfunction is the main player and early event, like scleroderma, lung fibrosis, or atherosclerosis. PMID:27083548

  17. Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria.

    Science.gov (United States)

    Gefen, Orit; Gabay, Chana; Mumcuoglu, Michael; Engel, Giora; Balaban, Nathalie Q

    2008-04-22

    Phenotypic variability in populations of cells has been linked to evolutionary robustness to stressful conditions. A remarkable example of the importance of cell-to-cell variability is found in bacterial persistence, where subpopulations of dormant bacteria, termed persisters, were shown to be responsible for the persistence of the population to antibiotic treatments. Here, we use microfluidic devices to monitor the induction of fluorescent proteins under synthetic promoters and characterize the dormant state of single persister bacteria. Surprisingly, we observe that protein production does take place in supposedly dormant bacteria, over a narrow time window after the exit from stationary phase. Only thereafter does protein production stop, suggesting that differentiation into persisters fully develops over this time window and not during starvation, as previously believed. In effect, we observe that exposure of bacteria to antibiotics during this time window significantly reduces persistence. Our results point to new strategies to fight persistent bacterial infections. The quantitative measurement of single-cell induction presented in this study should shed light on the processes leading to the dormancy of subpopulations in different systems, such as in subpopulations of viable but nonculturable bacteria, or those of quiescent cancer cells. PMID:18427112

  18. Conditional Cytotoxic Anti-HIV Gene Therapy for Selectable Cell Modification.

    Science.gov (United States)

    Garg, Himanshu; Joshi, Anjali

    2016-05-01

    Gene therapy remains one of the potential strategies to achieve a cure for HIV infection. One of the major limitations of anti-HIV gene therapy concerns recovering an adequate number of modified cells to generate an HIV-proof immune system. Our study addresses this issue by developing a methodology that can mark conditional vector-transformed cells for selection and subsequently target HIV-infected cells for elimination by treatment with ganciclovir (GCV). We used the herpes simplex virus thymidine kinase (TK) mutant SR39, which is highly potent at killing cells at low GCV concentrations. This gene was cloned into a conditional HIV vector, pNL-GFPRRESA, which expresses the gene of interest as well as green fluorescent protein (GFP) in the presence of HIV Tat protein. We show here that TK-SR39 was more potent that wild-type TK (TK-WT) at eliminating infected cells at lower concentrations of GCV. As the vector expresses GFP in the presence of Tat, transient expression of Tat either by Tat RNA transfection or transduction by a nonintegrating lentiviral (NIL) vector marked the cells with GFP for selection. In cells selected by this strategy, TK-SR39 was more potent at limiting virus replication than TK-WT. Finally, in Jurkat cells modified and selected by this approach, infection with CXCR4-tropic Lai virus could be suppressed by treatment with GCV. GCV treatment limited the number of HIV-infected cells, virus production, as well as virus-induced cytopathic effects in this model. We provide proof of principle that TK-SR39 in a conditional HIV vector can provide a safe and effective anti-HIV strategy. PMID:26800572

  19. Bezielle selectively targets mitochondria of cancer cells to inhibit glycolysis and OXPHOS.

    Directory of Open Access Journals (Sweden)

    Vivian Chen

    Full Text Available Bezielle (BZL101 is a candidate oral drug that has shown promising efficacy and excellent safety in the early phase clinical trials for advanced breast cancer. Bezielle is an aqueous extract from the herb Scutellaria barbata. We have reported previously that Bezielle was selectively cytotoxic to cancer cells while sparing non-transformed cells. In tumor, but not in non-transformed cells, Bezielle induced generation of ROS and severe DNA damage followed by hyperactivation of PARP, depletion of the cellular ATP and NAD, and inhibition of glycolysis. We show here that tumor cells' mitochondria are the primary source of reactive oxygen species induced by Bezielle. Treatment with Bezielle induces progressively higher levels of mitochondrial superoxide as well as peroxide-type ROS. Inhibition of mitochondrial respiration prevents generation of both types of ROS and protects cells from Bezielle-induced death. In addition to glycolysis, Bezielle inhibits oxidative phosphorylation in tumor cells and depletes mitochondrial reserve capacity depriving cells of the ability to produce ATP. Tumor cells lacking functional mitochondria maintain glycolytic activity in presence of Bezielle thus supporting the hypothesis that mitochondria are the primary target of Bezielle. The metabolic effects of Bezielle towards normal cells are not significant, in agreement with the low levels of oxidative damage that Bezielle inflicts on them. Bezielle is therefore a drug that selectively targets cancer cell mitochondria, and is distinguished from other such drugs by its ability to induce not only inhibition of OXPHOS but also of glycolysis. This study provides a better understanding of the mechanism of Bezielle's cytotoxicity, and the basis of its selectivity towards cancer cells.

  20. Organelle selection determines agonist-specific Ca2+ signals in pancreatic acinar and beta cells

    OpenAIRE

    Yamasaki, M.; Masgrau, R.; Morgan, A. J.; Churchill, G. C.; Patel, S.; Ashcroft, S. J. H.; Galione, A

    2004-01-01

    How different extracellular stimuli can evoke different spatiotemporal Ca2+ signals is uncertain. We have elucidated a novel paradigm whereby different agonists use different Ca2+-storing organelles ("organelle seleetion") to evoke unique responses. Some agonists select the endoplasmic reticulum (ER), and others select lysosome-related (acidic) organelles, evoking spatial Ca2+ responses that mirror the organellar distribution. In pancreatic acinar cells, acetylcholine and bombesin exclusively...

  1. Computational Image Analysis Reveals Intrinsic Multigenerational Differences between Anterior and Posterior Cerebral Cortex Neural Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Mark R. Winter

    2015-10-01

    Full Text Available Time-lapse microscopy can capture patterns of development through multiple divisions for an entire clone of proliferating cells. Images are taken every few minutes over many days, generating data too vast to process completely by hand. Computational analysis of this data can benefit from occasional human guidance. Here we combine improved automated algorithms with minimized human validation to produce fully corrected segmentation, tracking, and lineaging results with dramatic reduction in effort. A web-based viewer provides access to data and results. The improved approach allows efficient analysis of large numbers of clones. Using this method, we studied populations of progenitor cells derived from the anterior and posterior embryonic mouse cerebral cortex, each growing in a standardized culture environment. Progenitors from the anterior cortex were smaller, less motile, and produced smaller clones compared to those from the posterior cortex, demonstrating cell-intrinsic differences that may contribute to the areal organization of the cerebral cortex.

  2. Computational Image Analysis Reveals Intrinsic Multigenerational Differences between Anterior and Posterior Cerebral Cortex Neural Progenitor Cells.

    Science.gov (United States)

    Winter, Mark R; Liu, Mo; Monteleone, David; Melunis, Justin; Hershberg, Uri; Goderie, Susan K; Temple, Sally; Cohen, Andrew R

    2015-10-13

    Time-lapse microscopy can capture patterns of development through multiple divisions for an entire clone of proliferating cells. Images are taken every few minutes over many days, generating data too vast to process completely by hand. Computational analysis of this data can benefit from occasional human guidance. Here we combine improved automated algorithms with minimized human validation to produce fully corrected segmentation, tracking, and lineaging results with dramatic reduction in effort. A web-based viewer provides access to data and results. The improved approach allows efficient analysis of large numbers of clones. Using this method, we studied populations of progenitor cells derived from the anterior and posterior embryonic mouse cerebral cortex, each growing in a standardized culture environment. Progenitors from the anterior cortex were smaller, less motile, and produced smaller clones compared to those from the posterior cortex, demonstrating cell-intrinsic differences that may contribute to the areal organization of the cerebral cortex. PMID:26344906

  3. Systematic single-cell analysis of Pichia pastoris reveals secretory capacity limits productivity.

    Directory of Open Access Journals (Sweden)

    Kerry Routenberg Love

    Full Text Available Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.

  4. RhizoFlowCell system reveals early effects of micropollutants on aquatic plant rhizosphere.

    Science.gov (United States)

    Mynampati, Kalyan Chakravarthy; Lee, Yong Jian; Wijdeveld, Arjan; Reuben, Sheela; Samavedham, Lakshminarayanan; Kjelleberg, Staffan; Swarup, Sanjay

    2015-12-01

    In aquatic systems, one of the non-destructive ways to quantify toxicity of contaminants to plants is to monitor changes in root exudation patterns. In aquatic conditions, monitoring and quantifying such changes are currently challenging because of dilution of root exudates in water phase and lack of suitable instrumentation to measure them. Exposure to pollutants would not only change the plant exudation, but also affect the microbial communities that surround the root zone, thereby changing the metabolic profiles of the rhizosphere. This study aims at developing a device, the RhizoFlowCell, which can quantify metabolic response of plants, as well as changes in the microbial communities, to give an estimate of the stress to which the rhizosphere is exposed. The usefulness of RhizoFlowCell is demonstrated using naphthalene as a test pollutant. Results show that RhizoFlowCell system is useful in quantifying the dynamic metabolic response of aquatic rhizosphere to determine ecosystem health. PMID:26386206

  5. In vitro assessment of antiproliferative action selectivity of dietary isothiocyanates for tumor versus normal human cells

    Directory of Open Access Journals (Sweden)

    Konić-Ristić Aleksandra

    2016-01-01

    Full Text Available Background/Aim. Numerous epidemiological studies have shown beneficial effects of cruciferous vegetables consumption in cancer chemoprevention. Biologically active compounds of different Brassicaceae species with antitumor potential are isothiocyanates, present in the form of their precursors - glucosinolates. The aim of this study was to determine the selectivity of antiproliferative action of dietary isothiocyanates for malignant versus normal cells. Methods. Antiproliferative activity of three isothiocyanates abundant in human diet: sulforaphane, benzyl isothiocyanate (BITC and phenylethyl isothiocyanate, on human cervix carcinoma cell line - HeLa, melanoma cell line - Fem-x, and colon cancer cell line - LS 174, and on peripheral blood mononuclear cells (PBMC, with or without mitogen, were determined by MTT colorimetric assay 72 h after their continuous action. Results. All investigated isothiocyanates inhibited the proliferation of HeLa, Fem-x and LS 174 cells. On all cell lines treated, BITC was the most potent inhibitor of cell proliferation with half-maximum inhibitory concentration (IC50 values of 5.04 mmoL m-3 on HeLa cells, 2.76 mmol m-3 on Fem-x, and 14.30 mmol m-3 on LS 174 cells. Antiproliferative effects on human PBMC were with higher IC50 than on malignant cells. Indexes of selectivity, calculated as a ratio between IC50 values obtained on PBMC and malignant cells, were between 1.12 and 16.57, with the highest values obtained for the action of BITC on melanoma Fem-x cells. Conclusion. Based on its antiproliferative effects on malignant cells, as well as the selectivity of the action to malignant vs normal cells, benzyl isothiocyanate can be considered as a promising candidate in cancer chemoprevention. In general, the safety of investigated compounds, in addition to their antitumor potential, should be considered as an important criterion in cancer chemoprevention. Screening of selectivity is a plausible approach to the evaluation

  6. Dopamine Receptor Signaling in MIN6 β-Cells Revealed by Fluorescence Fluctuation Spectroscopy.

    Science.gov (United States)

    Caldwell, Brittany; Ustione, Alessandro; Piston, David W

    2016-08-01

    Insulin secretion defects are central to the development of type II diabetes mellitus. Glucose stimulation of insulin secretion has been extensively studied, but its regulation by other stimuli such as incretins and neurotransmitters is not as well understood. We investigated the mechanisms underlying the inhibition of insulin secretion by dopamine, which is synthesized in pancreatic β-cells from circulating L-dopa. Previous research has shown that this inhibition is mediated primarily by activation of the dopamine receptor D3 subtype (DRD3), even though both DRD2 and DRD3 are expressed in β-cells. To understand this dichotomy, we investigated the dynamic interactions between the dopamine receptor subtypes and their G-proteins using two-color fluorescence fluctuation spectroscopy (FFS) of mouse MIN6 β-cells. We show that proper membrane localization of exogenous G-proteins depends on both the Gβ and Gγ subunits being overexpressed in the cell. Triple transfections of the dopamine receptor subtype and Gβ and Gγ subunits, each labeled with a different-colored fluorescent protein (FP), yielded plasma membrane expression of all three FPs and permitted an FFS evaluation of interactions between the dopamine receptors and the Gβγ complex. Upon dopamine stimulation, we measured a significant decrease in interactions between DRD3 and the Gβγ complex, which is consistent with receptor activation. In contrast, dopamine stimulation did not cause significant changes in the interactions between DRD2 and the Gβγ complex. These results demonstrate that two-color FFS is a powerful tool for measuring dynamic protein interactions in living cells, and show that preferential DRD3 signaling in β-cells occurs at the level of G-protein release. PMID:27508444

  7. Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential.

    Directory of Open Access Journals (Sweden)

    Stéphane Bellafiore

    2008-10-01

    Full Text Available The root knot nematode, Meloidogyne incognita, is an obligate parasite that causes significant damage to a broad range of host plants. Infection is associated with secretion of proteins surrounded by proliferating cells. Many parasites are known to secrete effectors that interfere with plant innate immunity, enabling infection to occur; they can also release pathogen-associated molecular patterns (PAMPs, e.g., flagellin that trigger basal immunity through the nematode stylet into the plant cell. This leads to suppression of innate immunity and reprogramming of plant cells to form a feeding structure containing multinucleate giant cells. Effectors have generally been discovered using genetics or bioinformatics, but M. incognita is non-sexual and its genome sequence has not yet been reported. To partially overcome these limitations, we have used mass spectrometry to directly identify 486 proteins secreted by M. incognita. These proteins contain at least segmental sequence identity to those found in our 3 reference databases (published nematode proteins; unpublished M. incognita ESTs; published plant proteins. Several secreted proteins are homologous to plant proteins, which they may mimic, and they contain domains that suggest known effector functions (e.g., regulating the plant cell cycle or growth. Others have regulatory domains that could reprogram cells. Using in situ hybridization we observed that most secreted proteins were produced by the subventral glands, but we found that phasmids also secreted proteins. We annotated the functions of the secreted proteins and classified them according to roles they may play in the development of root knot disease. Our results show that parasite secretomes can be partially characterized without cognate genomic DNA sequence. We observed that the M. incognita secretome overlaps the reported secretome of mammalian parasitic nematodes (e.g., Brugia malayi, suggesting a common parasitic behavior and a possible

  8. Molecular Stratification of Clear Cell Renal Cell Carcinoma by Consensus Clustering Reveals Distinct Subtypes and Survival Patterns

    OpenAIRE

    Brannon, A. Rose; Reddy, Anupama; Seiler, Michael; Arreola, Alexandra; Moore, Dominic T.; Pruthi, Raj S.; Wallen, Eric M.; Nielsen, Matthew E; Liu, Huiqing; Nathanson, Katherine L.; Ljungberg, Börje; Zhao, Hongjuan; BROOKS, JAMES D.; Ganesan, Shridar; Bhanot, Gyan

    2010-01-01

    Clear cell renal cell carcinoma (ccRCC) is the predominant RCC subtype, but even within this classification, the natural history is heterogeneous and difficult to predict. A sophisticated understanding of the molecular features most discriminatory for the underlying tumor heterogeneity should be predicated on identifiable and biologically meaningful patterns of gene expression. Gene expression microarray data were analyzed using software that implements iterative unsupervised consensus cluste...

  9. Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin

    Directory of Open Access Journals (Sweden)

    Seok-Hyung Kim

    2011-03-01

    Tuberous sclerosis complex (TSC is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin or TSC2 (encodes tuberin genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242 in the tsc2 gene. This tsc2vu242 allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2vu242 is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2vu242/vu242 homozygous zebrafish, and is moderately increased in tsc2vu242/+ heterozygotes. Forebrain neurons are poorly organized in tsc2vu242/vu242 homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2vu242/vu242 mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

  10. Neutron scattering reveals extremely slow cell water in a Dead Sea organism

    OpenAIRE

    Tehei, Moeava; Franzetti, Bruno; Wood, Kathleen; Gabel, Frank; Fabiani, Elisa; Jasnin, Marion; Zamponi, Michaela; Oesterhelt, Dieter; Zaccai, Giuseppe; Ginzburg, Margaret; Ginzburg, Ben-Zion

    2007-01-01

    Intracellular water dynamics in Haloarcula marismortui, an extremely halophilic organism originally isolated from the Dead Sea, was studied by neutron scattering. The water in centrifuged cell pellets was examined by means of two spectrometers, IN6 and IN16, sensitive to motions with time scales of 10 ps and 1 ns, respectively. From IN6 data, a translational diffusion constant of 1.3 × 10−5 cm2 s−1 was determined at 285 K. This value is close to that found previously for other cells and close...

  11. High-throughput sequencing reveals an altered T cell repertoire in X-linked agammaglobulinemia

    OpenAIRE

    Ramesh, Manish; Simchoni, Noa; Hamm, David; Cunningham-Rundles, Charlotte

    2015-01-01

    To examine the T cell receptor structure in the absence of B cells, the TCR β CDR3 was sequenced from DNA of 15 X-linked agammaglobulinemia (XLA) subjects and 18 male controls, using the Illumina HiSeq platform and the ImmunoSEQ analyzer. V gene usage and the V–J combinations, derived from both productive and nonproductive sequences, were significantly different between XLA samples and controls. Although the CDR3 length was similar for XLA and control samples, the CDR3 region of the XLA T cel...

  12. Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry

    OpenAIRE

    Silva-Ayala, Daniela; López, Tomás; Gutiérrez, Michelle; Perrimon, Norbert; López, Susana; Arias, Carlos F.

    2013-01-01

    Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the en...

  13. Sulindac sulfide selectively increases sensitivity of ABCC1 expressing tumor cells to doxorubicin and glutathione depletion

    Science.gov (United States)

    Whitt, Jason D.; Keeton, Adam B.; Gary, Bernard D.; Sklar, Larry A.; Sodani, Kamlesh; Chen, Zhe-Sheng; Piazza, Gary A.

    2016-01-01

    Abstract ATP-binding cassette (ABC) transpo rters ABCC1 (MRP1), ABCB1 (P-gp), and ABCG2 (BCRP) contribute to chemotherapy failure. The primary goals of this study were to characterize the efficacy and mechanism of the non­steroidal anti-inflammatory drug (NSAID), sulindac sulfide, to reverse ABCC1 mediated resistance to chemother­apeutic drugs and to determine if sulindac sulfide can influence sensitivity to chemotherapeutic drugs independently of drug efflux. Cytotoxicity assays were performed to measure resistance of ABC-expressing cell lines to doxoru­bicin and other chemotherapeutic drugs. NSAIDs were tested for the ability to restore sensitivity to resistance selected tumor cell lines, as well as a large panel of standard tumor cell lines. Other experiments characterized the mechanism by which sulindac sulfide inhibits ABCC1 substrate and co-substrate (GSH) transport in isolated membrane vesicles and intact cells. Selective reversal of multi-drug resistance (MDR), decreased efflux of doxor­ubicin, and fluorescent substrates were demonstrated by sulindac sulfide and a related NSAID, indomethacin, in resistance selected and engineered cell lines expressing ABCC1, but not ABCB1 or ABCG2. Sulindac sulfide also inhibited transport of leukotriene C4 into membrane vesicles. Sulindac sulfide enhanced the sensitivity to doxoru­bicin in 24 of 47 tumor cell lines, including all melanoma lines tested (7-7). Sulindac sulfide also decreased intra­cellular GSH in ABCC1 expressing cells, while the glutathione synthesis inhibitor, BSO, selectively increased sensitivity to sulindac sulfide induced cytotoxicity. Sulindac sulfide potently and selectively reverses ABCC1-mediated MDR at clinically achievable concentrations. ABCC1 expressing tumors may be highly sensitive to the direct cytotoxicity of sulindac sulfide, and in combination with chemotherapeutic drugs that induce oxidative stress.

  14. Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer

    DEFF Research Database (Denmark)

    Su, Ying; Subedee, Ashim; Bloushtain-Qimron, Noga;

    2015-01-01

    heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for...

  15. Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching

    Science.gov (United States)

    Horns, Felix; Vollmers, Christopher; Croote, Derek; Mackey, Sally F; Swan, Gary E; Dekker, Cornelia L; Davis, Mark M; Quake, Stephen R

    2016-01-01

    Antibody class switching is a feature of the adaptive immune system which enables diversification of the effector properties of antibodies. Even though class switching is essential for mounting a protective response to pathogens, the in vivo patterns and lineage characteristics of antibody class switching have remained uncharacterized in living humans. Here we comprehensively measured the landscape of antibody class switching in human adult twins using antibody repertoire sequencing. The map identifies how antibodies of every class are created and delineates a two-tiered hierarchy of class switch pathways. Using somatic hypermutations as a molecular clock, we discovered that closely related B cells often switch to the same class, but lose coherence as somatic mutations accumulate. Such correlations between closely related cells exist when purified B cells class switch in vitro, suggesting that class switch recombination is directed toward specific isotypes by a cell-autonomous imprinted state. DOI: http://dx.doi.org/10.7554/eLife.16578.001 PMID:27481325

  16. Mucus-secreting 'signet-ring' cells in CSF revealing the site of primary cancer.

    OpenAIRE

    Agnelli, G.; Gresele, P.

    1980-01-01

    A case is reported of leptomeningeal carcinomatosis in which identification of mucus-secreting 'signet-ring' carcinoma cells in the CSF allowed diagnosis of an otherwise asymptomatic gastric cancer. When lumbar puncture is performed, careful cytological examination of the CSF should be carried out in any undiagnosed patient with neurological symptoms and signs.

  17. Dynamics between cancer cell subpopulations reveals a model coordinating with both hierarchical and stochastic concepts.

    Science.gov (United States)

    Wang, Weikang; Quan, Yi; Fu, Qibin; Liu, Yu; Liang, Ying; Wu, Jingwen; Yang, Gen; Luo, Chunxiong; Ouyang, Qi; Wang, Yugang

    2014-01-01

    Tumors are often heterogeneous in which tumor cells of different phenotypes have distinct properties. For scientific and clinical interests, it is of fundamental importance to understand their properties and the dynamic variations among different phenotypes, specifically under radio- and/or chemo-therapy. Currently there are two controversial models describing tumor heterogeneity, the cancer stem cell (CSC) model and the stochastic model. To clarify the controversy, we measured probabilities of different division types and transitions of cells via in situ immunofluorescence. Based on the experiment data, we constructed a model that combines the CSC with the stochastic concepts, showing the existence of both distinctive CSC subpopulations and the stochastic transitions from NSCCs to CSCs. The results showed that the dynamic variations between CSCs and non-stem cancer cells (NSCCs) can be simulated with the model. Further studies also showed that the model can be used to describe the dynamics of the two subpopulations after radiation treatment. More importantly, analysis demonstrated that the experimental detectable equilibrium CSC proportion can be achieved only when the stochastic transitions from NSCCs to CSCs occur, indicating that tumor heterogeneity may exist in a model coordinating with both the CSC and the stochastic concepts. The mathematic model based on experimental parameters may contribute to a better understanding of the tumor heterogeneity, and provide references on the dynamics of CSC subpopulation during radiotherapy. PMID:24416258

  18. Networks of neuroblastoma cells on porous silicon substrates reveal a small world topology

    KAUST Repository

    Marinaro, Giovanni

    2015-01-01

    The human brain is a tightly interweaving network of neural cells where the complexity of the network is given by the large number of its constituents and its architecture. The topological structure of neurons in the brain translates into its increased computational capabilities, low energy consumption, and nondeterministic functions, which differentiate human behavior from artificial computational schemes. In this manuscript, we fabricated porous silicon chips with a small pore size ranging from 8 to 75 nm and large fractal dimensions up to Df ∼ 2.8. In culturing neuroblastoma N2A cells on the described substrates, we found that those cells adhere more firmly to and proliferate on the porous surfaces compared to the conventional nominally flat silicon substrates, which were used as controls. More importantly, we observed that N2A cells on the porous substrates create highly clustered, small world topology patterns. We conjecture that neurons with a similar architecture may elaborate information more efficiently than in random or regular grids. Moreover, we hypothesize that systems of neurons on nano-scale geometry evolve in time to form networks in which the propagation of information is maximized. This journal is

  19. Targeted deletion of p73 in mice reveals its role in T cell development and lymphomagenesis.

    Directory of Open Access Journals (Sweden)

    Alice Nemajerova

    Full Text Available Transcriptional silencing of the p73 gene through methylation has been demonstrated in human leukemias and lymphomas. However, the role of p73 in the malignant process remains to be explored. We show here that p73 acts as a T cell-specific tumor suppressor in a genetically defined mouse model, and that concomitant ablation of p53 and p73 predisposes mice to an increased incidence of thymic lymphomas compared to the loss of p53 alone. Our results demonstrate a causal role for loss of p73 in progression of T cell lymphomas to the stage of aggressive, disseminated disease. We provide evidence that tumorigenesis in mice lacking p53 and p73 proceeds through mechanisms involving altered patterns of gene expression, defects in early T cell development, impaired apoptosis, and the ensuing accumulation of chromosomal aberrations. Collectively, our data imply that tumor suppressive properties of p73 are highly dependent on cellular context, wherein p73 plays a major role in T cell development and neoplasia.

  20. Long-range Transcriptome Sequencing Reveals Cancer Cell Growth Regulatory Chimeric mRNA

    Directory of Open Access Journals (Sweden)

    Roberto Plebani

    2012-11-01

    Full Text Available mRNA chimeras from chromosomal translocations often play a role as transforming oncogenes. However, cancer transcriptomes also contain mRNA chimeras that may play a role in tumor development, which arise as transcriptional or post-transcriptional events. To identify such chimeras, we developed a deterministic screening strategy for long-range sequence analysis. High-throughput, long-read sequencing was then performed on cDNA libraries from major tumor histotypes and corresponding normal tissues. These analyses led to the identification of 378 chimeras, with an unexpectedly high frequency of expression (≈2 x 10-5 of all mRNA. Functional assays in breast and ovarian cancer cell lines showed that a large fraction of mRNA chimeras regulates cell replication. Strikingly, chimeras were shown to include both positive and negative regulators of cell growth, which functioned as such in a cell-type-specific manner. Replication-controlling chimeras were found to be expressed by most cancers from breast, ovary, colon, uterus, kidney, lung, and stomach, suggesting a widespread role in tumor development.

  1. Dynamics between cancer cell subpopulations reveals a model coordinating with both hierarchical and stochastic concepts.

    Directory of Open Access Journals (Sweden)

    Weikang Wang

    Full Text Available Tumors are often heterogeneous in which tumor cells of different phenotypes have distinct properties. For scientific and clinical interests, it is of fundamental importance to understand their properties and the dynamic variations among different phenotypes, specifically under radio- and/or chemo-therapy. Currently there are two controversial models describing tumor heterogeneity, the cancer stem cell (CSC model and the stochastic model. To clarify the controversy, we measured probabilities of different division types and transitions of cells via in situ immunofluorescence. Based on the experiment data, we constructed a model that combines the CSC with the stochastic concepts, showing the existence of both distinctive CSC subpopulations and the stochastic transitions from NSCCs to CSCs. The results showed that the dynamic variations between CSCs and non-stem cancer cells (NSCCs can be simulated with the model. Further studies also showed that the model can be used to describe the dynamics of the two subpopulations after radiation treatment. More importantly, analysis demonstrated that the experimental detectable equilibrium CSC proportion can be achieved only when the stochastic transitions from NSCCs to CSCs occur, indicating that tumor heterogeneity may exist in a model coordinating with both the CSC and the stochastic concepts. The mathematic model based on experimental parameters may contribute to a better understanding of the tumor heterogeneity, and provide references on the dynamics of CSC subpopulation during radiotherapy.

  2. Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers.

    Science.gov (United States)

    Billing, Anja M; Ben Hamidane, Hisham; Dib, Shaima S; Cotton, Richard J; Bhagwat, Aditya M; Kumar, Pankaj; Hayat, Shahina; Yousri, Noha A; Goswami, Neha; Suhre, Karsten; Rafii, Arash; Graumann, Johannes

    2016-01-01

    Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC. PMID:26857143

  3. Single-cell sequencing reveals karyotype heterogeneity in murine and human malignancies

    NARCIS (Netherlands)

    Bakker, Bjorn; Taudt, Aaron; Belderbos, Mirjam E.; Porubsky, David; Spierings, Diana C. J.; de Jong, Tristan V.; Halsema, Nancy; Kazemier, Hinke G.; Hoekstra-Wakker, Karina; Bradley, Allan; de Bont, Eveline S. J. M.; van den Berg, Anke; Guryev, Victor; Lansdorp, Peter M.; Colome-Tatche, Maria; Foijer, Floris

    2016-01-01

    Background: Chromosome instability leads to aneuploidy, a state in which cells have abnormal numbers of chromosomes, and is found in two out of three cancers. In a chromosomal instable p53 deficient mouse model with accelerated lymphomagenesis, we previously observed whole chromosome copy number cha

  4. Selective abrogation of the uPA-uPAR interaction in vivo reveals a novel role in suppression of fibrin-associated inflammation

    DEFF Research Database (Denmark)

    Connolly, Brian M; Choi, Eun Young; Gårdsvoll, Henrik;

    2010-01-01

    overall structure of the domain. Analysis of Plau(GFDhu/GFDhu) mice revealed an unanticipated role of the uPA-uPAR interaction in suppressing inflammation secondary to fibrin deposition. In contrast, leukocyte recruitment and tissue regeneration were unaffected by the loss of uPA binding to uPAR. This...... study identifies a principal in vivo role of the uPA-uPAR interaction in cell-associated fibrinolysis critical for suppression of fibrin accumulation and fibrin-associated inflammation and provides a valuable model for further exploration of this multifunctional receptor....

  5. Comparative transcriptome analysis in induced neural stem cells reveals defined neural cell identities in vitro and after transplantation into the adult rodent brain

    Directory of Open Access Journals (Sweden)

    Anna-Lena Hallmann

    2016-05-01

    Full Text Available Reprogramming technology enables the production of neural progenitor cells (NPCs from somatic cells by direct transdifferentiation. However, little is known on how neural programs in these induce